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Microbe-Host Relationships in Immunology Poster Abstracts

June 9, 2017 Simon Fraser University

Harbour Centre

2017

POSTER #1 Elucidating the mechanism behind sex difference in asthma prevalence

Laura Mathae1,2 and Fumio Takei1,3

1Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC, Canada 2Interdiscpinlary Oncology Program, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada 3Department of

Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada

Epidemiological studies have shown higher prevalence of asthma in women compared to men during reproductive age. Although sex steroid hormones (SSH) are suspected to be responsible, the mechanism is unknown. Asthma is caused by chronic inflammation of the airways, which occurs due to excessive type 2 immune responses mediated by type 2 T helper cells and group 2 innate lymphoid cells (ILC2s). ILC2s are activated by epithelial-derived cytokines IL-33, IL-25 and thymic stromal lymphopoietin (TSLP) and produce type 2 cytokines, IL-5 and IL-13. IL-5 induces eosinophil infiltration, while IL-13 causes mucus hyperproduction, leading to allergic inflammation. We have previously identified ILC2s in mouse lung and recently found that ILC2s in female mice proliferate more than those in male littermates upon intranasal (i.n) IL-33 injections and in vitro stimulation by IL-33 and TSLP. The sex bias is age dependent as it is only present in pubertal mice, but not in younger mice. Time-course analyses of ILC2s and eosinophils demonstrated that the sex bias occurs early during inflammation. In vitro cultures of purified ILC2s with SSH revealed that testosterone may inhibit ILC2 proliferation and/or function, while estrogen had little effect. Interestingly, female ILC2s exhibit a more activated gene expression profile compared to male ILC2s. These results suggest that exposure to SSH may induce intrinsic changes in ILC2s, which make female ILC2s more prone to activation and proliferation compared to male ILC2s.

POSTER #2 Activation of memory Group 2 Innate Lymphoid Cells

Itziar Martinez-Gonzalez1,2, Maryam Ghaedi2,3, Laura Mathae2,3, Catherine A. Steer2,3,

Fumio Takei1,2

1Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, B.C., Canada.

2Terry Fox Laboratory British Columbia Cancer Agency, Vancouver, B.C., Canada. 3Interdisciplinary Oncology Program, University of British Columbia, Vancouver, B.C., Canada.

In response to allergens, Group 2 Innate Lymphoid Cells (ILC2s) produce type 2 cytokines, which induce acute allergic lung inflammation. ILC2s do not directly recognize allergens but they are stimulated by alarmins released by the damaged epithelium. Some of the previously activated ILC2s persist as memory cells and respond to unrelated allergens more potently than naïve ILC2s, mediating severe allergic inflammation. Memory ILC2s acquire the ability to respond to the alarmin IL-25. However, preliminary data suggest that co-stimulatory signals are required. Understanding how memory ILC2s are regulated will lead to the development of new therapies for allergic lung diseases.

POSTER #3

The carboxyl tail of connexin43 influences BCR signaling-induced cytoskeletal rearrangements in B-Lymphocyte

Farnaz Pournia 1,2, Kate Choi 1,2, May Dang-Lawson2 and Linda Matsuuchi1,2 1CELL and Developmental Biology Graduate Program, 2Department of Zoology, University of British Columbia

Cytoskeletal-dependent cellular processes such as adhesion, membrane spreading, immune synapse formation, and migration are crucial for normal B cell development and for immune responses. Signaling by the B cell antigen receptor (BCR), integrins and chemokine receptors results in Rap-dependent actin remodeling. We have previously shown that Connexin43 (Cx43) is important for B cell adhesion, spreading, motility and directed migration and that the carboxyl tail (CT) of Cx43 is critical for these effects, in particular the region of the CT between amino acids 246-307 that contains a number of key tyrosine (Y) and serine (S) residues. Using a mutational approach, and the cell spreading assay as a convenient readout to assess cytoskeletal rearrangements, the effects of individual Cx43 point mutants or combinations of mutants was studied. Our analysis of the serine mutants, S255A, S262A, S279A and S282A showed that they had differential effects on B cell spreading. We hypothesized that this reflected unique roles for these serine residues in leading to actin remodeling. Future studies will include identifying the proteins that interact with the Cx43 CT at these key sites and how these interactions could influence the dynamics of the actin network. This may reveal critical interactions that are nucleated by Cx43 and link receptors to molecular mechanisms that control cellular cytoskeletal architecture that influence B Lymphocyte development and immune responses.

POSTER #4

The role of the Rap1 GTPase and cofilin in mast cell activation Caitlin Pritchard, Kate Choi, May Dang-Lawson, Michael R. Gold

Department of Microbiology and Immunology, University of British Columbia

Mast cells are central players in allergic and inflammatory responses but the signaling pathways leading to mast cell activation are not fully understood. The major activating receptor on mast cells is the Fcε Receptor I (FcεRI), a cell surface receptor that binds the constant region of IgE antibodies. The binding of multivalent antigens to IgE-FcεRI complexes initiates intracellular signaling cascades that lead to mast cell effector functions that are responsible for allergic disease. Some of these responses are highly dependent on actin remodelling; for example, degranulation relies on localized actin clearance to allow fusion of cytoplasmic granules with the plasma membrane and phagocytic cup formation depends on controlled actin rearrangement. Cofilin, an actin severing protein, is known to be activated by FcεRI signaling. However, the mechanism leading to cofilin-mediated actin remodeling and the role of cofilin in effector functions in mast cells is not fully known. Our lab has previously shown that the Rap1 GTPase (Rap1) is a master regulator of actin dynamics and organization in B cells by activating cofilin. Our hypothesis is that Rap1 plays an essential role in controlling actin-dependent FcεRI-mediated effector functions in mast cells by inducing actin remodelling through cofilin. Using immunoblotting, we have found that Rap1 is activated by FcεRI signaling in mouse bone marrow-derived mast cells. Loss-of-function approaches will be used to determine whether Rap1 activation is essential for the activation of cofilin and to determine the importance of both proteins in actin-dependent mast cell responses. This work will generate a better understanding of the mechanisms underlying mast cell activation signaling and may identify new drug targets for allergy treatment and prevention.

POSTER #5

Systems level mass cytometric analysis of the effects of tumour-derived cytokines on myeloid cell development and function in a pre-clinical

model of breast cancer. Israel Matos, Maunish Barvalia, Alex Sio, Manreet Cheval, Kenneth Harder

Department of Microbiology and Immunology, University of British Columbia,

Vancouver, British Columbia, Canada. Cancer immunotherapies have improved patient outcome and survival in a subset of human malignancies. However, particular types of tumours and a significant number of patients do not respond to current therapies, highlighting the need to better understand the interactions between tumours and the immune system. Phagocytes such as dendritic cells (DCs), macrophages and myeloid-derived suppressor cells (MDSCs) are critical in orchestrating or antagonizing innate and adaptive immune responses against tumours. In turn, phagocyte activity and development is heavily dependent on tumour-derived factors.

We have identified a tumour-derived factor that alters phagocyte development and function. We hypothesize that this cytokine perturbs the myeloid compartment by impairing phagocyte development and maturation, resulting in tumour-induced immunosuppression. Our results show that this tumour-derived factor impairs DC maturation and induces the expansion of immature myeloid cell progenitors and MDSCs. Interestingly, the cytokine is particularly powerful in terms of its ability to block the development of a subset of DCs (XCR1+CD11b-), which has been associated with improved cancer treatment outcome and strong CD8+

cytotoxic T cell activation potential. Our in vivo data show that this tumour-secreted factor leads to anemia, leukocytosis, splenomegaly and enhances extramedullary hematopoiesis. Finally, we present cytometry by time-of-flight (CyTOF) data showing that tumour growth, and its associated cytokine expression, leads to a systemic accumulation of a complex mixture of immature myeloid cells, with associated perturbations in a wide variety of signalling pathways throughout the immune system. We believe that modulating this tumour-secreted factor could be key to improve immunotherapy efficacy.

POSTER #6

Cellular metabolic and translational constraints in preterm monocytes

Christina Michalski1,2, Bernard Kan1,2, Hilda HT Au3, Dan Luciani1,4, Colin Ross1,5, Eric Jan3, Pascal M. Lavoie1,2,6

1BC Children’s Hospital’s Research Institute, Vancouver; 2Department of Medicine or 3Biochemistry and Molecular Biology or 4Surgery or 6Pediatrics, or 5Faculty of Pharmaceutical Sciences, UBC, Vancouver

Human innate immune responses are broadly attenuated below 32 weeks of gestation, resulting in increased infections in infants born prematurely. Recently, it has been shown that innate immune cells require a metabolic switch from oxidative phosphorylation to aerobic glycolysis, in order to supply the massive energy requirement for protein synthesis during an immune activation. This switch is regulated through the mechanistic target of rapamycin (mTOR) master immune regulator. However, the underlying mechanism(s) controlling the activity of innate immune cells in humans during ontogeny remain unclear. We hypothesized that the broad functional attenuation in preterm neonatal immune cells is due to a metabolic reprogramming resulting in decreased translation capacity of immune response proteins. To address this hypothesis, we combined genome-wide transcriptome analyses (Illumina HT-12 Human Bead Array) and polysome profiling (qPCR) comparing preterm, term (cord) and adult (peripheral) primary (blood) monocytes after stimulation with LPS (endotoxin). We found that LPS-stimulated (5h) preterm monocytes express a wide array of cytokine and chemokine gene transcripts, without expression of the corresponding proteins. In pathway analyses, we identify metabolic disturbances in these cells. This was confirmed by showing a reduced lactate production and reduced glycolytic capacity in these cells, as measured using real-time extracellular acidification (Agilent Seahorse Analyzer). We also found that genes encoding ribosomal subunits were severely down-regulated both before, but also after stimulation in preterm monocytes, compared to term or adult monocytes. Our preliminary data suggests that translation is substantially reduced in preterm monocytes (as demonstrated by radioactive Met/Cys pulse-labeling), and that this mechanism is mTOR-related as evidence by lack of mTOR phosphorylation in preterm monocytes. Through polysome profiling experiments we demonstrate a relationship between the developmental lack of immune signaling proteins and their degree of translation. Altogether, our data provide a novel mechanism through which immune activation is constrained during fetal life.

POSTER #7

Renal Outcomes in Pediatric Anti-neutrophil Cytoplasmic Antibody Associated Vasculitis – A Pediatric Vasculitis Initiative (PEDVAS) Study

A. Chen1, C. Mammen1,2, D. Cabral1,3, K.L. Brown1,3 and K. Morishita1,3 for the

ARChiVe and PedVas Network of investigators

1Faculty of Medicine, University of British Columbia 2Division of Pediatric Nephrology, BC Children’s Hospital 3Division of Pediatric Rheumatology, BC Children’s Hospital

Objectives: Renal disease is the most common complication affecting pediatric patients with ANCA-associated vasculitis (AAV). However, renal outcomes have not been well described in children with AAV. Our objectives were to describe the early renal disease course and 12-month outcomes in pediatric AAV. Methods: From an international registry (ARCHiVE) consisting of patients from 40 sites, patients diagnosed with AAV <18 years of age with pauci-immune glomerulonephritis on renal biopsy or a decrease in renal function requiring dialysis were included. Patients’ renal findings and investigations were examined at presentation and at 12 months following presentation. Glomerular filtration rate (GFR) based outcomes included the proportion of children with normal GFR (>90 ml/min/1.73m2), moderately reduced (MR) GFR (30-60 ml/min/1.73m2), severely reduced (SR) GFR (15-30 ml/min/1.73m2) and renal failure (<15 ml/min/1.73m2). GFR was estimated using the Schwartz equation. Results: Of the 71 patients who met inclusion criteria, 69% were female, 57% were Caucasian, and 82% had GPA (granulomatosis with polyangiitis). Initial GFR of the 63 patients with creatinine data at presentation were as follows: renal failure (32%, n=20), SR-GFR (14%, n=9), MR-GFR (22%, n=14) and normal GFR (32%, n=20). Renal findings of the 71 patients on presentation include hypertension (37%), oliguria (23%) and nephrotic syndrome (17%). 27% of patients required dialysis at presentation. At 12 months, 64% of those requiring dialysis at presentation either remained on dialysis (n=7) or received a kidney transplant (n=2). Only 38% of patients at 12 months had a normal GFR while 41% and 3% had MR- and SR-GFR respectively. Conclusion: Early renal disease in pediatric AAV is often severe with over two thirds of patients presenting with a GFR<60 ml/min/1.73m2 and one quarter of patients requiring acute dialysis. Even though a number of patients improved after induction therapy, only 38% of patients have a normal GFR at 12-months. Future analysis from the ARCHIVE registry will allow us to better explore longer term outcomes as well as predictors of outcomes.

POSTER #8

Age-Associated B cells: A Proposed Mechanism of Viral Contribution to Multiple Sclerosis

Isobel Mouat, Iryna Shanina, Ana Citlali Marquez, Jessica Allanach, and Marc Horwitz

Department of Microbiology and Immunology, University of British Columbia

Though the origins have proven elusive, inflammation has been shown to cause heterogeneous damage in Multiple Sclerosis (MS) patients. It has been proposed that age-associated B cells (ABCs) are a probable culprit of inflammation in MS. ABCs are a recently defined pro-inflammatory B cell subset that expands with age particularly in females, and are associated with viral infection and autoimmunity. Following stimulation with viral TLR7/9 ligands, ABCs preferentially secrete proinflammatory molecules IFNg and IL10, and undergo class switch from IgM to pathogenic IgG2a/c. ABCs arise earlier in autoimmune-prone mice and are present in higher frequencies in autoimmune patients. Depletion of ABCs results in decreased autoantibody production in a mouse model of lupus. Thus, it is likely that the expansion of ABCs and subsequent inflammation is a mechanism by which viral infection, specifically Epstein-Barr Virus (EBV), contributes to MS.

Our research group has developed a model of MS in which mice are infected with murine herpesvirus 68, a homolog of EBV, prior to enhanced autoimmune encephalomyelitis (EAE) induction. The resulting Th1/Th17 immunological profile more closely resembles that observed in MS patients compared to traditional EAE. In this model, heightened numbers of ABCs are observed in infected mice, and correlate with both viral load and clinical severity. Further, phenotypic differences in antigen presentation and cytokine production between ABCs isolated from infected versus uninfected mice have been identified through transplantation experiments.

The elucidation of the contributions of B cell subsets to MS is particularly appealing due to the success of B cell-depletion therapies. In conclusion, the inflammation resulting from the expansion of ABCs is a potential mechanism by which EBV contributes to MS pathology.

POSTER #9

Disruption of the Gut Microbiota with Antibiotics Increases Acute Vascular Rejection

K. Rey, S. Manku, W. Enns, A. von Rossum, J.C. Choy

Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada

Background: Immunological damage of blood vessels in heart transplants contributes to acute rejection by causing arteritis, which is characterized by excessive immune cell infiltration and damage of medial smooth muscle cells, and chronic rejection, which is characterized by arteriosclerotic thickening of the intima. The endogenous gut microbiota influences many immunological processes but whether its disruption affects immune responses that lead to graft rejection is not known.

Methods: A model of vascular rejection involving the interposition grafting of a segment of abdominal aorta from a Balb/c donor mouse into the infrarenal aorta of an allogeneic C57Bl/6 recipient mouse was used. The gut microbiota was disrupted in graft recipients starting as neonates using an antibiotic cocktail (ampicillin, vancomycin, metronidazole, neomycin sulfate) in their drinking water.

Results: Ablation of gut bacteria with antibiotics led to a significant loss of medial area, reflective of increased acute rejection, but no difference in intimal thickening, which reflects chronic rejection. Antibiotic treatment for only the first three weeks of life was sufficient to increase acute rejection. Arteries from antibiotic treated recipients also showed more early infiltration of CD4 T cells, CD8 T cells, and neutrophils as compared to untreated counterparts. Further, treatment of graft recipients with antibiotics led to greater production of IFNγ, IL-17 and IL-4 from T cells after transplantation, and this was related to reduced expression of Foxp3, indicative of fewer T regs.

Conclusion: Disruption of the gut microbiota with antibiotics alters allogeneic immune responses and increases the development of acute vascular rejection.

POSTER #10 Expression and regulation of amphiregulin in human regulatory T cells

Avery J Lam1, Sabine M Ivison1, Anne M Pesenacker1, Guy Charron2, Haiming

Huang3, iGenoMed Consortium, Sachdev S Sidhu3, James G Pan3, John D Rioux2, Megan K Levings1

1Department of Surgery, University of British Columbia and BC Children’s Hospital Research Institute;

2Montréal Heart Institute and Université de Montréal; 3Toronto Recombinant Antibody Centre, University of Toronto

Regulatory T cells are essential for maintaining immune homeostasis and self-tolerance. Several lines of evidence suggest that adoptive transfer of Tregs can prevent, and in some cases cure, a variety of pathological conditions, from autoimmunity to transplant rejection. In addition to their effects on immune cells, there is also emerging evidence that Tregs have direct effects on tissue repair. Specifically, Tregs in mice promote tissue repair after infection or injury by producing the EGF family member amphiregulin (AREG) under the control of the alarmins IL-18 and IL-33. We investigated expression and regulation of AREG in human peripheral blood Tregs. TCR stimulation of Tregs (CD4+CD25hiCD127lo) increased AREG mRNA and protein, particularly in the HLA-DR- subset, but levels were lower than in their Tconv counterparts. In contrast to reports from murine Tregs, IL-18 and IL-33 did not modulate human Treg production of AREG. Ex vivo Tregs expressed IL-18Rα, but expression of IL-33Rα (ST2) was not detectable. To more accurately measure human ST2 expression, we used phage display to generate a series of anti-ST2 mAbs. Experiments in ST2-transfected HEK-293T and transduced T cells revealed several candidate anti-ST2 mAbs that were superior to commercially available mAbs for flow cytometric detection of human ST2. Experiments to better define the localization and biology of human ST2+ Tregs are in progress. Knowledge of whether human Tregs produce AREG is important to understand their potential to mediate tissue repair in addition to immunosuppression when used as a cell-based therapy.

POSTER #11 Probing primary immune responses to prospective immunogen designs

aimed at triggering VRC01-class neutralizing antibodies to HIV-1

Alekhya Josyula Venkata1 and Ralph Pantophlet1,2

1Department of Molecular Biology and Biochemistry and 2Faculty of Health Sciences, SFU

A major goal of current HIV vaccine research is the design of immunogens to elicit broadly active virus-neutralizing antibodies (bnAbs). VRC01 is prototypic for a group of ~30 bnAbs that all derive from the IGHV1-2*02 gene. These bnAbs bind the conserved CD4-binding site on HIV gp120, which is an attractive target for immunogen design. Wildtype recombinant gp120 or other recombinant forms of the HIV spike typically do not yield bnAbs, including those of the VRC01 class, which may be attributable to distracting Ab epitopes and poor engagement of the proper naïve B cells in vivo. We are therefore exploring a gp120 design strategy encompassing modifications to engage VRC01-class precursors derived from naïve B cells and removal of unwanted Ab epitopes or occlusion with added glycans. The V3 loop on gp120 harbors unwanted Ab epitopes but also potentially important CD4 T-cell epitopes that, if eliminated, could disrupt adequate T-cell help. Here, we investigated changes in primary immune responses to a gp120-derived antigen with a glycan-masked V3 and appended with a copy of a pan-DR T-helper epitope (‘PADRE’). Two other antigens, one without any of the modifications and one with only the glycan-masked V3, were used as comparators. Tfh and germinal center (GC) B cell frequencies in inguinal lymph nodes of C57BL/6 mice (n=5/group) primed with antigen adjuvanted in Quil-A were assessed on day 7 and frequencies of bone marrow Ab-secreting cells (ASCs) and splenic memory B cells on day 28. Antigen-specific serum Ab titers were assessed on days 7 and 28 by ELISA. We found Tfh frequencies to be significantly and equally decreased in both animal groups given antigen with a glycan-masked V3 compared to animals immunized with antigen without the added modifications, indicating that the single PADRE motif was insufficient to rescue T-cell help. Accordingly, GC B cell frequencies were also substantially lower in these animals and antigen-specific Abs not detectable in ELISA. Unexpectedly, antigen-specific memory B cell and ASC frequencies in animals given unmodified antigen were found to be as low as those in the other two animal groups despite robust Tfh induction. These results suggest the possible necessity to consider both the level and quality of Tfh responses in immunogen design strategies to prime and properly boost VRC01-class bnAb responses.

POSTER #12 Releasable RapidSpheres™ enable immunomagnetic purification of

highly viable and functional immune cells from complex tissues in less than 30 minutes

Jennifer Kennett, Alisse Saunders, Samuel J. Clarke, Andy I. Kokaji, Drew W. Kellerman, Catherine Ewen, Martina N. Chambers, Mandy Chan and Steven M.

Woodside

STEMCELL Technologies Inc. Vancouver, BC, Canada. Recent advances in cellular therapy and diagnostics promise dramatic improvements in disease prevention and treatment. This research is driven by scientists studying how cells of the immune system communicate and relies on the success of cell-based assays. However, it remains challenging and time-consuming to purify large numbers of high-quality cells from complex tissues. EasySep™ Release is a fast and easy cell isolation method which utilizes the Releasable RapidSpheres™. This novel magnetic particle technology improves cell purities by reducing contaminants and allows for gentle particle removal to mitigate potential interference in downstream assays. The 29-minute protocol involves first incubating target cells with antibody complexes followed by the Releasable RapidSpheres™. Next, labelled cells are purified using a hand-held magnet. Particle-free purified cells are obtained by applying a mild dissociation reagent and a final magnetic separation. We validated EasySep™ Release by purifying T, B and NK cells from peripheral blood mononuclear cell samples containing 5 to 800 million cells. Cell purities were 99.7 ± 0.1% (n=17) for CD3+ T cells, 99.4 ± 0.4% (n=17) for CD4+ cells, 98.8 ± 0.5% (n=17) for CD8+ T cells, 98.6 ± 0.7% (n=6) for CD19+ cells and 95.6 ± 1.7% (n=6) for CD56+ cells. Isolated cells were confirmed particle-free, viable and functional. We obtained similar high performance using unprocessed leukapheresis samples of up to 5 billion cells, demonstrating excellent scalability and compatibility with more complex samples. Finally, we show how EasySep™ Release can be paired with commercially available antibodies or sequential separations to isolate almost any cell type, including those with a complex phenotype.

POSTER #13 Improving T Cell Trafficking to Tumors by Modulating the Resident

Memory (Trm) Phenotype

Julian Smazynski1, 2, John R Webb1, Victoria Hodgson1, Brad H Nelson1, 2

Trev and Joyce Deeley Research Centre, BC Cancer Agency1; Department of Biochemistry and Microbiology,

University of Victoria, Victoria BC2

Adoptive Cell Transfer (ACT) is a personalized therapy which involves extracting T cells from tumors, followed by in vitro expansion and reinfusion; however, major challenges exist in promoting the infiltration of T cells into solid tumors. Intratumoral CD8+ T cells expressing the tissue resident memory (Trm) marker CD103 strongly associate with patient survival. CD103 is the integrin αEβ7 and is upregulated on T cells by co-stimulation with antigen and TGFβ. By binding to E-cadherin, CD103 promotes localization of T cells to epithelial tissues. This raises the possibility that T cells expressing Trm features may demonstrate altered infiltration properties in solid epithelial cancers. Murine CD8+ T cells were expanded in vitro with TGFβ; as a negative control, T cells are cultured with an inhibitor of TGFβR1 (LY2157299). T cells were adoptively transferred into tumor-bearing mice to determine the trafficking of cells in vivo. Tumors were analyzed via flow cytometry and monitored over time to assess therapeutic efficacy. In vitro experiments have shown that compared to T cells expanded in the presence of TGFβ, T cells cultured with LY2157299 showed negligible expression of CD103, indicating reversal of the Trm phenotype. In preliminary adoptive transfer experiments, LY2157299-cultured OT-I cells showed increased numbers in peripheral blood and greater anti-tumor efficacy compared to TGFβ-cultured CD103+ OT-I. These experiments suggest that inhibition of TGFβ during expansion can establish a CD103 negative phenotype with enhanced therapeutic efficacy. Modulating the Trm phenotype may represent an effective means to produce TIL products with improved therapeutic potency, thus improving ACT.

POSTER #14

IL-6 expression is correlated with increased T cell proliferation and survival in the arterial wall in Giant Cell Arteritis

Sukhbir Manku 1, Wendy Wong 2, Zongshu Luo 1, Zainab Alabdurubalnabi 2, Michael A. Seidman 3, Kevin Rey 1, Winnie Enns 1, J. Antonio Avina-Zubieta 2,4, Kamran

Shojania 2,4, Jonathan C. Choy 1*

1 Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada 2 Arthritis Research Canada, Richmond, British Columbia, Canada 3 Department of Pathology and

Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada 4 Division of Rheumatology, University of British Columbia, Vancouver, British Columbia, Canada

Objective: Giant cell arteritis (GCA) is the most common vasculitis in adults affecting large and medium-sized arteries. IL-6 and T cell accumulation within the arterial wall contribute to the pathogenesis of GCA, and recent clinical studies have shown the blockade of IL-6 activity is efficacious in its treatment. We examined the relationship between levels of IL-6 and immunological processes that control the expansion of T cells within GCA lesions. Methods: The expression of IL-6 RNA was quantified by RT-qPCR and the frequency of proliferating CD4 T cells, T regs, and T cells undergoing apoptotic cell death quantified by immunohistochemical staining in GCA-positive temporal artery biopsies. Results: CD4 T cells accumulated in clusters within the media and deep intima of all GCA lesions. There was a significant positive correlation between the expression of IL-6 and increased frequency of proliferating CD4 T cells (r = 0.76, p < 0.01). The expansion of T cells can be inhibited by T regs but IL-6 expression was not correlated with differences in T reg accumulation. Increased IL-6 levels were also significantly correlated with lower frequencies of CD4 T cells undergoing apoptotic cell death (r = -0.81, p < 0.01). Conclusion: IL-6 may contribute to the accumulation of CD4 T cells in GCA by supporting their proliferation and survival within the arterial wall through mechanisms that are independent of effects on local T reg expansion.

POSTER #15 Fucose availability and its utilization impact in vivo enteric pathogen

virulence.

JM Allaire, K Bhullar, SM Crowley, M Stahl, H Yang, EF Verdu, HB Yu, BA Vallance

Department of Pediatrics, BC Children’s Hospital Research Institute, Vancouver

Attaching and effacing (A/E) bacterial pathogens such as Enterohemorrhagic E. coli (EHEC) are important causes of diarrheal disease worldwide. However, little is known about how these bacteria acquire key nutrients from the host, and whether these nutrients modulate pathogen colonization and infection of the gut. For example, fucose is an important sugar and bacterial food source cleaved from host glycans found on the intestinal mucosal surface via the actions of commensal microbes expressing fucosidase enzymes. Interestingly, although the mouse A/E pathogen Citrobacter rodentium does not itself express a fucosidase, recent studies have found that it and EHEC use a fucose sensing system to modulate their pathogenicity in vitro. At present, it is unclear what role fucose metabolism may play in A/E pathogen virulence and metabolism in vivo. Aim: We investigated the roles of fucose and the enzyme L-fuculose kinase in controlling C. rodentium pathogenesis. Methods: Wildtype (WT) and ΔfucK C. rodentium were used for several in vitro assays (type three secretion, growth and adhesion assays) and for in vivo infection. C57Bl/6 mice were infected either separately, or with both C. rodentium strains at a dose of 10^8 colony forming units (CFU). In some infections, mice were pretreated with streptomycin (20mg, 24h before infection) by oral gavage, and/or fed with fucose (200ul of 25mM L-fucose 2x/day). Bacterial burdens, pathology score and competitive assay were assessed on day 6 post-infection (6 DPI). Results: In vitro virulence assays identified no significant differences between ΔfucK and WT C. rodentium. Moreover, both WT and ΔfucK C. rodentium readily colonized the intestines of mice either pretreated (or not), with streptomycin, with pathogen burdens, localisation and histological pathology scores similar between the two strains. In contrast, simultaneous infection by both strains (competitive fitness assay) within the same mice revealed the ΔfucK strain was significantly impaired when competing with WT C. rodentium (6 DPI, no streptomycin). In contrast, ΔfucK was able to equally compete with WT C. rodentium when streptomycin pretreatment was given to deplete commensal bacteria. Conclusion: These results indicate that the fucK-dependent fucose metabolic pathway promotes (but is not essential to) C. rodentium pathogenesis. Moreover these findings suggest that commensal microbes play a key role in controlling fucose availability within the gut, and thereby impact A/E pathogen metabolism and virulence.

POSTER #16 Statistical and Functional Analyses Reveal Subtype-specific Restrictions

on HIV Adaptation to Host Cellular Immunity

Natalie Kinloch1§, G Lee2§, J Carlson3, C Brumme2, H Byakwaga4,5, C Muzoora4, K Cobarrubias1, M Brockman1,2, P Hunt5, J Martin5, M Carrington6, D Bangsberg7, PR

Harrigan2,8, Zabrina Brumme1,2

1 Faculty of Health Science, SFU 2 BC CfE in HIV/AIDS 3 Microsoft Research 4 Mbarara University of Science and Technology, Uganda 5 UC San Francisco 6 Frederick National Laboratory for Cancer Research, Frederick, MD 7 MGH and Harvard University, Boston, MA 8 Department of Medicine, UBC § denotes equal contribution

Background: The extent to which viral genetic context constrains HLA-driven immune escape pathways in HIV remains incompletely understood. We combine phylogenetically-informed statistical analyses with in vitro functional assessments to investigate HLA-driven adaptation in a host population where multiple HIV-1 subtypes co-circulate (Uganda). Methods: HLA-associated polymorphisms in HIV-1 Gag, Pol and Nef were identified in 200 antiretroviral-naive individuals infected with subtype A1 and 135 infected with subtype D. Their strengths of selection were then compared across HIV-1 subtypes using a phylogenetically-informed logistic regression approach to identify instances of differential selection between subtypes. Multiple testing was addressed using q-values. Infectious molecular clones expressing consensus and mutant subtype A1 or D gag/protease sequences in an HIV-1 NL4.3 backbone were constructed using Gibson Assembly and used to produce VsVg-pseudotyped virus stocks. In vitro replication of these viruses was assessed using a 7-day GFP-reporter-based assay. Results: A total of 83 Gag, 198 Pol and 105 Nef HLA-associated polymorphisms were identified in subtype A1 and/or D at q<0.2 (all p<9x10-4). Of these, 34% (Gag), 39% (Pol) and 27% (Nef) exhibited significant differential selection between subtypes (p<0.05; q<0.1). For example, HLA-B*57:03 strongly selected Gag-T242N in subtype D (Odds Ratio [OR]=250; p=2x10-10), but not subtype A1 (OR=1.8; p=0.8)(inter-subtype comparison p=8x10-6; q=0.001). This raised the hypothesis that the subtype A1 consensus proline at adjacent Gag codon 243, which differs from the consensus leucine observed in subtype D, is incompatible with T242N. Indeed, a subtype A1 virus carrying 242N/P243 exhibited >10-fold poorer in vitro replication compared to consensus A1, confirming HIV-1 subtype-specific constraints on immune escape at this position. Conclusions: Statistical analyses applied to linked HIV-1/HLA datasets can illuminate HIV-1 codons where mutational immune escape pathways may be constrained in certain HIV-1 subtypes. Functional validation of these incompatible mutation combinations may help identify subtype-specific mutationally-constrained viral regions for vaccine design.

POSTER #17 Antigen Presentation by Dendritic Cell Subsets Promote or Protect Autoimmune Diabetes Onset Following Coxsackievirus B Infection

Zachary Morse, Iryna Shanina, Dr. Marc Horwitz

University of British Columbia Department of Microbiology and Immunology

Genetic inheritance is only partially responsible for determining onset of the autoimmune disease, type 1 diabetes (T1D). Epidemiological and clinical evidence, as well as discordance rates between monozygotic twins in regards to T1D development indicate environmental factors such as virus infections, vitamin D deficiency, dietary antigens, and gut dysbiosis also play a significant part in disease development. Genome-wide association studies have found several mutations in specific gene loci which confer greater susceptibility to T1D, including those involved in virus recognition, immune signaling, and antigen presentation. The most prominent etiological agent thought to trigger T1D onset is the enterovirus, coxsackievirus B (CVB). Our lab has shown that reducing the expression of the dsRNA viral receptor, melanoma differentiation-associated protein 5 (MDA5), in NOD mice can effectively prevent susceptibility to T1D onset following CVB4 infection. Mice with reduced MDA5 exhibit a prominent type I interferon (IFN) profile and regulatory T cell response following virus infection to prevent autoimmunity. However, there are indications that type III IFN also contribute to the diabetogenic response. Subsequent antiviral actions of APCs have a significant role in priming an individual for development of an autoreactive adaptive response by skewing toward a more proinflammatory state. While the secretion of type I IFN by APCs is largely thought to promote the onset of autoimmunity; dendritic cell activation and antigen presentation via expression of MHC I and II may indicate important mechanisms that lead to autoreactivity against the insulin-secreting beta cells of the pancreas following virus infection.

POSTER #18 CD44 and Inflammation Induced Hyaluronan in Tumor Metastasis.

1Arif A., 2Freeman S.A., 2Austin P., Gilmour M., 1Poon G., 1Dong Y., 1Gold M.,

1Johnson P., & 2Roskelley C.D.

1Department of Microbiology and Immunology, 2Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver V6T 1Z3

Invading tumor cells leave the primary tumor and enter peripheral organs where they encounter new environments. The cytokines, cells, and extracellular matrix (ECM) encountered there are important in determining success in forming a secondary lesion, and inflammation provides changes for all these factors that may create a pro-metastatic environment. Hyaluronan (HA) is an ECM component that is dynamically regulated during inflammation and binds to the cell surface protein CD44. CD44 is expressed on tumor and host cells, and is known to mediate functions on immune cells. The effect of CD44 and HA in the metastatic environment, and specifically, if and how they function in mediating tumor-immune cell interactions during inflammation will be investigated. The B16F0 melanoma model of lung metastasis is used, where cells are injected I.V. into inflamed lungs and assayed at late and early time points. We find that bleomycin (BLM) induced inflammation induces HA deposition in the lungs, and a large influx of differentiating macrophages. Injection of B16F0 cells at the peak of HA in BLM results in increased metastatic nodules in the lung. B16 cells isolated form the lungs are increased in BLM and are induced to bind HA. Furthermore, CS was found to be inhibitory to HA binding on CD44 as removal of total cell surface CS or inhibition of CS addition on CD44 resulted in constitutive high HA binding. Together, these results will help us understand the roles of HA binding and its regulation by CS modification in tumor metastasis, as well as shed light on HA mediated tumor-host cell interactions.

POSTER #19

Development of GMP-compatible protocols for thymus-derived regulatory T cell expansion

Katherine MacDonald1,2, Romy Hoeppli3#, James Piret2,4, Esme Dijke5,6,#, Lori

West5,6,7,#, Megan Levings2#

1Department of Biomedical Engineering, University of British Columbia, Vancouver, BC, Canada 2Michael

Smith Laboratories, University of British Columbia, Vancouver, BC, Canada 3Department of Surgery, University of British Columbia, Vancouver, BC, Canada 4Department of Chemical and Biological Engineering,

University of British Columbia, Vancouver, BC, Canada 5Department of Pediatrics, University of Alberta, Edmonton, AB, Canada 6Alberta Transplant Institute, University of Alberta, Edmonton, AB, Canada

7Department of Surgery, University of Alberta, Edmonton, AB, Canada #Canadian National Transplant Research Program Investigator

Animal and early clinical studies have shown that regulatory T cell (Treg) therapy can prevent graft-versus-host disease (GVHD) following hematopoietic stem cell transplant. We have previously found that the thymus, which is routinely removed during pediatric cardiac surgery, is a plentiful source of Tregs. However, to use this therapy in humans, GMP-compliant methods for isolation and large-scale expansion of thymic Tregs must be developed. CD4+CD8-CD25+ thymic Tregs were isolated from pediatric thymuses using GMP-compatible magnetic bead-based separation. Tregs were expanded using artificial antigen-presenting cells or GMP-compatible antibody tetramer complexes that stimulate CD3 and CD28, with or without additional activation of CD2. Thymic Tregs expand in vitro when cultured with tetrameric antibody complexes. About 50-fold expansion was achieved using the anti-CD3/CD28/CD2 antibody tetramers, which represents the expansion that will be necessary to reach a clinically relevant dose. A higher expansion rate was obtained using the anti-CD3/CD28/CD2 antibody tetramer complexes than the anti-CD3/CD28. Cells cultured with these antibody tetramers express high levels of FOXP3, similar levels of Treg-characteristic markers as those expanded with artificial antigen-presenting cells, and do not produce inflammatory cytokines. Thymic Tregs can be isolated and expanded with GMP-compliant methods. Once the protocol is optimized, we can transfer our protocols to a GMP facility for further process development and to generate data required for a clinical trial application.

POSTER #20 Vasoactive Intestinal Peptide Promotes Th17 Immune Responses

Thereby Protecting Against Citrobacter rodentium Infection

HB YU1, X Wu1, H Yang1, ES Bosman1, FA Graef1, LS Celiberto1, C Ma1, T Huang1, JC Berkmann1, A Fotovati2, GS Reid2, JA Waschek3, LC Osborne4, BA Vallance1, K,

Jacobson1

1Division of Gastroenterology, 2Division of Oncology, Department of Pediatrics, BCCHR and UBC, 3David

Geffen School of Medicine, University of California, 4Department of Microbiology & Immunology, UBC Vasoactive intestinal peptide (VIP), a 28 amino acid neuropeptide, exhibits potent anti-inflammatory effects in autoimmune and inflammatory diseases. This is though to be due to its ability to inhibit Th1 (proinflammatory) cell responses as well as promote Th2 (anti-inflammatory) cell responses. However, recent ex vivo studies suggest that VIP can also promote the production of Th17 T cells (primarily secrete interleukine IL-17). While Th17 cells augment inflammation in autoimmune diseases, they are also known to protect against extracellular pathogens such as Citrobacter rodentium, a model organism to study the pathogenesis of two important human pathogens, enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC). We hypothesized that VIP deficient mice (Vip-/-) would be more susceptible to C. rodentium due to defective Th17 cell responses. Indeed, Vip-/- mice infected with C. rodentium showed dramatic weight loss, lower survival rates, increased pathogen colonization at intestinal and systemic sites, worsened mucosal damage/inflammation as well as reduced numbers of IL-17 producing T cells. We also found that the exaggerated tissue damage seen in Vip-/- mice was driven by a functional type III secretion system, a critical virulence program encoded by C. rodentium. Importantly, exogenous treatment of Vip-/- mice with recombinant VIP ameliorated C. rodentium-induced colitis, accompanied by restoration of IL-17 production. Collectively, we provide the first in vivo evidence that VIP protects against C. rodentium infection through the promotion of Th17 immune responses. It also highlights that the interactions between the immune and enteric nervous systems can play an important role in modulating the outcomes of infectious diseases.

POSTER #21 EasySep™ kits for immunomagnetic purification of CD138+ cells from

mouse and rat samples Trevor Rogers1, Kathleen MacDonald1, Whalley Fong1, Peter Repenning1, Alexisann

Maxwell1, Mark Brown1, Terry Thomas1, and Allen Eaves1,2

1STEMCELL Technologies Inc., and 2The Terry Fox Laboratory, Vancouver, BC, Canada

CD138 is found on several B cell subsets but is highly expressed on antibody-secreting cells (plasma cells and plasmablasts). Because CD138+ antibody-secreting cells represent a small fraction of the total B cell population (0.4 ± 0.1% (n = 52), 1.6 ± 1.1% (n = 43), and 0.6 ± 0.5% (n = 18) when using naïve C57BL/6 mice, immunized BALB/c mice, or naïve rat (Sprague Dawley and Wistar) spleens, respectively), pre-enrichment of CD138+ cells can improve downstream assays and processes such as hybridoma screening. Therefore, we have developed a facile immunomagnetic method to isolate CD138+ cells from mouse and rat samples. The column-free immunomagnetic method allows for isolation of CD138+ cells in less than 25 minutes from mouse or rat spleen, bone marrow, and other tissues using fast and simple EasySep™ technology. Bispecific antibody complexes are used to cross-link CD138+ cells to dextran-coated magnetic particles and the labeled cells are separated from the unwanted cells using an EasySep™ magnet. Single-cell suspensions of splenocytes and bone marrow cells do not require red blood cell lysis prior to isolation. Using this method, the total viable CD138+ content of the isolated fraction is typically 75.0 ± 10.2% (n = 41), 77.9 ± 1.6% (n = 4), and 55.5 ± 10.3% (n = 52), when using naïve C57BL/6 mice, immunized BALB/c mice, or naïve rat spleens, respectively, while the high-expressing CD138+ antibody-secreting cell content of the isolated fraction is typically 49.5 ± 16.2% (n = 41), 71.6 ± 3.4% (n = 4), and 48.6 ± 10.8% (n = 52), respectively. The isolated CD138+ populations remain functional and are immediately ready for downstream applications, including antibody development projects.

POSTER #22 Role of phospholipid targeting antibodies in anti-tumor mediated

immunity in colorectal cancer

Allison McNamara, Desmond Pink, Courtney Mowat and Kristi Baker

University of Alberta, Faculty of Medicine and Dentistry, Department of Oncology, Edmonton Alberta Colorectal cancer (CRC) patients have a poor prognosis and targeted therapy is difficult due to lack of conserved tumor associated antigens (TAA). The majority of CRC have a limited response to immunotherapy due in part to the highly tolerant nature of the mucosal immune system. Immunotherapies for CRC directed at TAA that can overcome intestinal tolerance need to be developed to improve the survival of CRC patients. Exosomes secreted by CRC can carry a bolus of TAA and display unique phospholipids (PPL) not typically found on the outer leaflet of the parental cell. These unique PPL can be bound by immunoglobulin G (IgG) to generate immune complexes (IC) and we hypothesize that engagement of the Fc region of the IC with Ig-binding receptors on dendritic cells (DC) can cross-prime CD8+ T-cell mediated CRC targeted immunity strong enough to overcome mucosal immunosuppression. Exosomes were isolated from mouse serum or tissue culture supernatant and flow analysis indicated that serum exosomes were bound by low levels of endogenous IgG but that PPL binding sites were not saturated since coating with anti-PPL resulted in high IgG binding. Stimulation of GM-CSF matured bone marrow derived DC with serum exosomes coated or not in extra anti-PPL resulted in increased expression of inflammatory cytokines especially where extra anti-PPL was present. This suggests that exosomes could stimulate strong immune responses when complexed with anti-PPL IgG. Further investigation of anti-PPL will allow us to better understand whether this strategy could help increase anti-tumor immunity and survival in CRC patients. Research funded by support from Queen Elizabeth the II Masters Student Scholarship and Supervisor Dr. Kristi Baker

POSTER #23 Regulation of IL-6 Signaling in Human Vascular Endothelial Cells by TNF

Ashani Montgomery, Kevin Rey, Ankana Mukherjee and Jonathan C. Choy

Department of Molecular Biology and Biochemistry, Simon Fraser University

Heart transplantation is the only therapeutic option for patients with end-stage heart failure. One of the main contributors to chronic rejection of heart transplants is transplant arteriosclerosis (TA), a vascular disease characterized by intimal hyperplasia and vasomotor dysfunction of graft arteries that develops as a result of the host immune response targeting arteries in the graft. Successful prevention of TA requires elucidation of the mechanisms involved in immune targeting and injury of allograft arteries. Interleukin -6 (IL-6), an inflammatory cytokine secreted within hours of surgical transplantation, plays an important role in the development of TA by supporting the activation of immune cells and preventing persistent injury of the luminal endothelium. Understanding how IL-6 signaling is regulated in vascular endothelial cells will be important for the application of therapeutic strategies that inhibit IL-6. I have been interested in determining how IL-6 signaling in vascular endothelial cells is affected by other inflammatory cytokines. TNF is a central inflammatory cytokine that is abundant early after organ transplantation where it coincides with IL-6 expression. Unstimulated human vascular endothelial cells do not express detectable levels of IL-6 receptor but are responsive to IL-6 trans-signaling induced by IL-6 and soluble IL-6R, a complex that then binds to the gp130 signaling subunit on the cell surface. Stimulation of endothelial cells with TNF reduces IL-6 trans-signaling in endothelial cells and this is related to the down-regulation of gp130 from the cell surface, potentially due to internalization. In conclusion, human vascular endothelial cells respond to IL-6 through trans-signaling and this is inhibited by TNF.

POSTER #24 CD44 and hyaluronan binding negatively regulate memory formation by

CD8 effector T cells

Sally S. M. Lee-Sayer1, Nina Maeshima1, Meghan Dougan1,2, Anita Dahiya1,2, Arif Arif1, Manisha Dosanjh1, Chris Maxwell2 and Pauline Johnson1

1 Department of Microbiology and Immunology, UBC 2 Department of Pediatrics, British Columbia Children’s

Hospital Research Institute, UBC

Effector and memory CD8 T cell formation is influenced by various cells and factors, but the role of the extracellular matrix in CD8 memory formation is poorly understood. Hyaluronan is a component of the extracellular matrix, and upon activation, CD8 T cells up-regulate the hyaluronan receptor CD44 and a subset of cells is induced to bind hyaluronan. Here, we determined that the extent of induced hyaluronan binding correlated with the peptide affinity and avidity for the TCR, and high affinity/ avidity interactions promoted the highest percentage of hyaluronan-binding cells. Interaction with hyaluronan has been linked to effector T cell proliferation and cell death in previous studies, suggesting a role for CD44 and hyaluronan in regulating effector T cell lifespan. To investigate the role of CD44 and hyaluronan binding in the CD8 T cell response, an equal mixture of CD44+/+ and CD44-/- OT-I CD8 T cells were adoptively transferred into congenic hosts, which were then intravenously infected with ovalbumin-expressing Listeria monocytogenes. While CD44+/+ and CD44-/- cells formed equal numbers of effector T cells, CD44-/- cells out-competed CD44+/+ cells in the generation of memory cells in the lymph nodes and bone marrow, and this competitive advantage was observed after the contraction phase. This demonstrated that CD44 is a negative regulator of high affinity OT-I CD8 T cell memory formation. When in competition, hyaluronan-binding CD8 effector T cells were found to have increased pAkt expression and glucose uptake, both of which positively regulate CD8 T effector proliferation and negatively regulate memory potential. This suggested that hyaluronan promotes effector CD8 T cells and reduces their ability to make the metabolic switch required for memory formation. Furthermore, hyaluronan-binding CD8 effector T cells had increased caspase 3 and 7 activity and showed increased death, indicating a preference for these cells to be lost during the contraction phase. Overall, this work implicates CD44 and hyaluronan binding as negative regulators of survival through contraction and memory formation by high affinity OT-I CD8 T cells.

POSTER #25 The Role of MALT1 in Macrophages

Mahdis Monajemi, Susan C. Menzies, and Laura M. Sly

BC Children’s Hospital Research Institute and the University of British Columbia At BC Children’s Hospital, a patient with severe combined immunodeficiency accompanied by dramatic inflammation along the gastrointestinal tract was diagnosed with a homozygous mutation in the intracellular signalling molecule, MALT1. MALT1 plays two distinct roles within cells: 1) It acts as a scaffolding protein required for Nuclear Factor kappa B (NFκB) activation, 2) It has protease activity, which can enhance or inhibit NFκB-driven transcription, depending upon its target. The consequences of MALT1 immunodeficiency have largely been attributed to its role in T and B cells, in which MALT1 acts downstream of the T or B cell receptor to drive lymphocyte proliferation and activation. MALT1 is also activated in macrophages downstream of the C-type lectin receptors, dectin-1 and dectin-2, and toll-like receptor 4 (TLR4). However, the role of MALT1 in macrophage-mediated inflammation has not been explored. Intestinal inflammation is largely driven by macrophages, suggesting that macrophage Malt1 may contribute to this inflammation. Based on this, we hypothesize that Malt1 deficiency in macrophages causes intestinal inflammation by increasing inflammatory cytokine production. We have found that Malt1 deficient murine macrophages actually have lower inflammatory cytokine production than wild type macrophages. In contrast, pharmacological inhibition of Malt1 protease activity increases inflammatory cytokine production in response to innate immune stimulation. In vivo, Malt1 deficiency (Malt1-/-) exacerbates DSS-induced colitis in mice. However, depletion of macrophages only modestly protects Malt1 deficient mice against DSS-induced colitis. Furthermore, adoptive transfer of macrophages does not change the susceptibility of Malt1-/- mice to DSS-induced colitis. Taken together, our studies are consistent with a model in which MALT1 activity reduces pro-inflammatory macrophage responses, but its scaffolding function increases macrophage inflammatory responses. In future studies, we will investigate the cell-specific contribution of MALT1 deficient macrophages to inflammatory disease using mice with myeloid-specific MALT1 deficiency. These studies will provide critical information about the cell specific role of MALT1 and possible side effects of MALT1 inhibitors currently used for lymphoma treatment.

POSTER #26

Memory B cells latently infected with γHV-68 generate a localized interferonopathy that acts to drive autoimmunity and facilitate Multiple

Sclerosis

Ana Citlali Márquez, Iryna Shanina and Marc S. Horwitz

Department of Microbiology and Immunology, The University of British Columbia, Vancouver, British Columbia, Canada, V6T 1Z3

Epstein-Barr virus (EBV) has been identified as a putative trigger of multiple sclerosis (MS). We hypothesize that latent EBV infection alters the immune response to an unknown second stimulus thereby exacerbating autoimmunity. Previously, we reported that mice latently infected with murine gammaherpesvirus 68 (γHV-68), the murine homolog to EBV, were induced for experimental autoimmune encephalomyelitis (EAE), they developed an enhanced disease more reminiscent of MS. Upon establishment of latency in memory B cells, a persistent upregulation of CD40 on dendritic cells accompanied by STAT1 phosphorylation was observed without infection of dendritic cells (DCs). Further, expression of CD40 on DCs in latently infected mice was required to prime the strong TH1 response driving disease as well as decrease Treg frequencies in the periphery and CNS. Latent gammaherpesvirus infection leaves a long-lasting immune impact that enhances subsequent immune responses that direct enhanced autoimmunity even after 5 months of latent infection. To maintain latency, EBV and γHV-68 require interferon-a (IFNα) expression. Localized increases in IFNα promote the strength and polarity of the adaptive T cell response. Chronic expression of IFNα promotes memory T cell activation and fine-tunes the antigenic specificity of the T cell while downregulating Tregs. In response to the presence of latent virus infection, memory B cells likely act as a strong source of Type I IFN response that alters the adjacent immune cell community of the lymph node (LN). It is our contention that EBV and γHV-68 create a localized interferonopathy at the level of the lymph node and, when EAE or another environmental triggers encounter the LN, a strong poorly regulated autoreactive Th1 response is generated. This represents a novel mechanism by which EBV can influence MS and other autoimmune diseases. To test this hypothesis, we examined our model in mice deficient for the IFNα receptor (IFNARko) as well as in mice depleted of CD8 T cells.

POSTER #27

Understanding the Sequence Requirements of Host Defence Peptides: From Antibiofilm Activity to Immune Modulation

Evan F. Haney1, Yoan Brito-Sanchez2, Michael J. Trimble1, Ashley L. Hilchie1, Sarah

C. Mansour1, Art Cherkasov2 and Robert E.W. Hancock1

1Centre for Microbial Diseases and Immunity Research, UBC. 2Prostate Centre at Vancouver General Hospital, UBC.

Host defence peptides (HDPs) are polypeptide sequences found ubiquitously in nature that have garnered significant attention as alternatives to antibiotics. In addition to their direct antibacterial effect, some HDPs possess antibiofilm activity or the ability to modulate the immune response of the host. Curiously, HDPs with potent activity in one arena are often weakly active with respect to other activities described for this class of molecules. In principle, synthetic HDPs could be optimized for specific biological purposes provided sufficient sequence information were available to predict the activity of novel peptides. Unfortunately, beyond the generic properties of HDPs (eg. positive charge and amphipathicity), our current understanding of the peptide sequence requirements that govern these diverse biological effects is limited, particularly for the immunomodulatory and antibiofilm properties. To overcome this challenge, it is necessary to screen large peptide libraries for their antibiofilm and immunomodulatory properties and to relate the differences in primary amino acid sequence to the observed differences in biological activities. Using a synthetic HDP as a sequence template (IDR-1018, VRLIVAVRIWRR-NH2), a substitution library of 96 single amino acid variants was SPOT-synthesized on cellulose membranes and the biological activity profile of each derivative was evaluated in vitro. Specifically, the antibiofilm efficacy against Gram-positive methicillin resistant Staphylococcus aureus (MRSA) as well as the immunomodulatory activity towards peripheral blood mononuclear cells was examined. The data from these in vitro screens were used to establish, for the first time, quantitative structure activity relationship (QSAR) models that identified important molecular descriptors that govern the antibiofilm and immunomodulatory characteristics of synthetic peptides. These QSAR models were subsequently used to predict the activities of virtual peptides which identified novel HDPs with unique biological activity profiles. Overall, this work improves our understanding of the sequence requirements of HDPs and paves the way for designing synthetic HDPs for specific therapeutic applications.

POSTER #28

Induction of Human Regulatory T Cells by a Parasite-Derived TGF-β Mimic

Laura Cook1, Brett de Bie1, Qing Huang3, May Q Wong1, Jana K Gillies3, Danielle J

Smyth2, Rick M Maizels2 and Megan K Levings3

1Department of Medicine, University of British Columbia, Vancouver, BC, Canada 2Wellcome Trust Centre for

Molecular Parasitology, University of Glasgow, Glasgow, UK 3Department of Surgery, University of British Columbia, Vancouver, BC, Canada

Immune homeostasis in the intestinal tract is tightly controlled by FOXP3+ regulatory T cells (Tregs); loss of this Treg-mediated control is linked to inflammatory bowel disease (IBD). As a mechanism of immune evasion, several species of intestinal parasites can strengthen intestinal Treg activity, leading to the notion that parasite-derived products could be harnessed and used as immunoregulatory therapy for IBD. It has been previously demonstrated that H. polygyrus, a natural murine parasite, produces a protein which mimics the ability of mammalian TGF-β to induce expression of FOXP3 in CD4+ T cells and promote intestinal immune regulation in vivo. Distinct from mammalian TGF-β, however, this TGF-β mimic (TGM) does not appear to have pro-fibrotic effects. Our aim was to determine whether TGM promotes the development of human FOXP3+ Tregs. Human naïve CD45RA+CD4+CD25– T cells were stimulated with anti-CD3/CD28 mAbs in the presence of TGM or TGF-β. The cells were analyzed over time for adoption of a Treg phenotype, as judged by FOXP3 expression, and de-methylation of the Treg-specific demethylated region (TSDR) of FOXP3. We found that TGM efficiently induced FOXP3 expression (>50%) in human naïve CD4+ T cells, and caused epigenetic modification of the TSDR to a greater extent than TGF-β indicative of stable lineage differentiation. We next asked if TGM could also convert potentially pathogenic memory T cells into Tregs. Human Th1, Th2 and Th17 cells were sorted ex vivo, then stimulated in the presence or absence of TGM or TGF-β. Surprisingly we found that TGM (and to a lesser extent TGF-β) strongly promoted the conversion of Th1 and Th17, but not Th2 cells, into FOXP3+ cells which acquired regulatory function and lost methylation at the TSDR. These data indicate that TGM has potential to be used to generate Tregs to treat IBD, and via its ability to convert Th1 and Th17 cells into regulatory cells, may have a unique capacity to reverse harmful pro-inflammatory effects of these cells.

POSTER #29

Neonatal type 2 biased lung immunity is shaped by group 2 innate lymphoid cells

Catherine A. Steer1,2, Itziar Martinez-Gonzalez1, Maryam Ghaedi1,3, Patricia Allinger1, 4, Laura Mathä1,2, Fumio Takei1,3

1Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, Canada 2Interdisciplinary Oncology Program, Faculty of Medicine, University of British Columbia, Vancouver, Canada 3Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada 4University of Applied Sciences,

Vienna, Austria Neonatal immunity is functionally distinct from adults as responses to environmental antigens are predominantly T helper (Th)-2 (type 2) biased. The neonatal lung is introduced to airborne antigens soon after birth, and early exposure has been linked to development of airway allergic diseases. However, our knowledge of neonatal lung immunity is limited and why it is type 2 biased is unknown. Our lab has characterized group 2 innate lymphoid cells (ILC2s) in mouse lungs, which upon exposure to inhaled allergens, produce IL-5 and IL-13 and drive type 2 lung inflammation. Here we investigated whether ILC2s play a role in neonatal type 2 bias and allergen sensitization. Mouse lung ILC2s rapidly developed and 10 -13 day old (D10-D13) pups had more ILC2s than adults. D10 ILC2s are activated due to high intracellular IL-13 and IL-5 expression and eosinophil lung infiltration, which were not observed in ILC2-deficient pups. ILC2s may be driving Th2-biased cell differentiation, as intranasal (IN) injection of OVA into D10 ILC2-deficient pups, transplanted with adult OT-II T cells, results in significantly more Th2 cells in the lung-draining lymph node (LN) after 6 days compared to wildtype mice. Furthermore, upon IN protease-allergen papain at D10 and 4 weeks later, higher numbers of LN Th2 cells were present compared to in mice without neonatal papain. Il33-/- D10 pups had fewer activated ILC2s and little eosinophil lung infiltration, suggesting that neonatal ILC2s are activated by endogenous IL-33 resulting in type 2-biased lung immunity. Interestingly, neonatal, not adult, ILC2s respond more intensely to a viral infection mimic, poly I:C (IN injection). As neonatal viral infections are known to lead to allergy development later in life, ILC2s appear to play a pivotal role in shaping a window of opportunity for type 2 biased lung immunity, increasing the risk of allergen sensitization in the neonatal period.

POSTER #30

Integrative Analysis of Multi-Omic Data in Sepsis

Samuel Hinshaw1,2, Amy Lee1, Robert Hancock1

1Centre for Microbial Diseases and Immunity Research, University of British Columbia, Vancouver, BC, V6T

1Z4 2Graduate Program in Bioinformatics, BCCA 100-570 West 7th Avenue, Vancouver,BC, V5Z 4S6

The integration and analysis of data from a variety of “omics” platforms is a major goal in the bioinformatics community. While RNA-Seq transcriptomics provides a comprehensive picture of gene expression, other omics data, especially metabolomics, is relatively sparse. Metabolomics profiling elucidates the chemical products of pathways but does not directly report on the events that gave rise to those products. Methods allowing these data to be integrated would provide a more comprehensive picture of cellular function, from signalling to metabolic output. However, integration of these disparate data types has proven difficult.

Here we present a novel network-based integrative analysis method using disparate data sets from pediatric and adult patients with septic shock. Specifically, we combined comprehensive RNA-Seq gene expression profiling with relatively-sparse NMR metabolomics. Metabolites were linked to their anabolic and catabolic enzymes, and from these enzymes, we generated first-order metabolic networks using experimentally-validated protein-protein interactions. These networks demonstrated significant functional overlap with zero-order protein-protein interaction networks generated from transcriptomics data, indicating that both omics technologies report on similar biological processes. By creating a combined network from both datasets, we identified genes of interest through comparative hub analysis. Overall, this methodology shows promise for application to multi-omics studies of diseases, specific stimuli, and therapies.

POSTER #31

Homing Capacity of Thymic Regulatory T Cells Can Be Tailored by Expansion in Cytokine-enriched Culture Conditions

Romy E Hoeppli1,5, Esmé Dijke2,3,5, Andrew IM Campbell1, Lori J West2,3,4,5 and Megan

K Levings1,5

1Surgery, University of British Columbia, Vancouver, British Columbia, Canada; 2Pediatrics, University of Alberta, Edmonton, Alberta, Canada; 3Alberta Transplant Institute, University of

Alberta, Edmonton, Alberta, Canada; 4Surgery, University of Alberta, Edmonton, Alberta, Canada and 5Canadian National Transplant Research Program, Canada.

Regulatory T cell (Tregs)-based therapy is a promising approach to treat allograft rejection. We have previously found that thymuses, routinely removed during pediatric cardiac surgery, are a potential source of therapeutic Tregs. To be effective, Tregs must express homing receptors for migration to inflammatory sites. For example, expression of the chemokine receptor CXCR3 on Tregs is essential to guide Tregs to locations of Th1-inflammation. We hypothesized that migration of thymic Tregs to Th1-inflammation could be fine-tuned by including cytokines in the expansion protocol. CD4+CD25+ Tregs were isolated from pediatric thymuses and expanded with artificial antigen-presenting cells, rapamycin and IL-2. IL-12 and IFN-gamma were included for Th1-polarizing conditions. Thymic Tregs cultured under Th1-polarizing conditions significantly increased CXCR3 and T-bet expression and showed >2-fold higher expansion capacity compared to Tregs cultured in neutral conditions. Expression of CXCR3 persisted even after removal of the polarizing cytokines. Importantly, Tregs cultured with Th1-inducing cytokines maintained a stable phenotype, including high FOXP3 expression and low TSDR methylation. Levels of other Treg-associated markers remained unchanged between neutrally and Th1-cultured Tregs at RNA- and protein-level. Th1-polarized Tregs did not acquire the ability to produce Th1-cell associated cytokines such as IL-2 or IFN-gamma and potently suppressed proliferation of total conventional T cells and Th1-effector T cells in vitro. In contrast to neutral cultures, expansion under Th1-conditions enabled thymic Tregs to migrate towards the CXCR3-specific chemokine CXCL10 in vitro. Thus, expansion conditions of thymic Tregs can be manipulated to specifically and stably tailor the cells’ homing capacity. The ability to direct Tregs towards specific tissues or sites of inflammation may enable optimal targeting as a therapeutic in vivo. This means that Tregs could act in a more specific manner which would reduce pan-immunosuppression often associated with polyclonal Treg administration in animal models and early clinical trials.

POSTER #32

Depletion of Dead Cells from Primary Tissue in 6 Minutes Christine Barr, Karina McQueen1, Carrie E. Peters1, Steven M. Woodside1, Terry E.

Thomas1 , Allen C. Eaves1,2. 1STEMCELL Technologies Inc, Vancouver, BC, Canada; 2BC Cancer Agency, Vancouver, BC, Canada

Tissue specific research often requires enzymatic digestion to isolate and study certain cell types. The digestion can be a harsh process that results in a lot of dead cells in the final single cell suspension. Subsequent analysis by flow cytometry is difficult to interpret due to non-specific binding of antibodies to dead cells and dead cell auto fluorescence. Factors released by dead cells can also interfere with downstream assays, complicating the study of many primary tissues.

During apoptosis, the cell membrane loses its energy-dependent phospholipid asymmetry, resulting in exposure of negatively charged phospholipids on the cell surface. Relocation of phosphatidylserine (PS) to the outer leaflet of the cell membrane is a well-established marker of apoptosis. We have developed a rapid method (EasySep™) to remove dead cells from a primary tissue sample. Briefly, immunomagnetic depletion of dead cells is achieved by targeting exposed PS with Annexin V along with an antibody complex that links Annexin V to magnetic particles. The cell suspension is then placed in an EasySep™ magnet and live cells are poured or pipetted off and ready for use in as little as 6 minutes.

Performance of this kit was examined on various mouse and human tissue types. Using this method, we were able to improve viability of a single cell suspension of mouse lungs digested with collagenase/hyaluronidase from a start viability of 39.6 ± 12.3% AnxV-/PI- to 70.9 ± 11.8% AnxV-/PI. From 1x10^8 total start cells, 1.43 ± 0.68 x10^7 live cells were recovered (n=10). From human polymorphonuclear leukocytes (PMN) cultured overnight, viability was improved from 23.7 ± 9.8% AnxV-/PI- to 67.7 ± 12.1% AnxV-/PI- with recovery of 1.27 ± 0.52 x10^7 live cells from 1x10^8 total start cells (n=7). This equates to removal of 88.9 ± 8.3% and 91.8 ± 5.3% of dead cells respectively. Since live cells are untouched, subsequent isolation of desired cell types can be performed, resulting in a more viable population of cells for downstream applications.

POSTER #33

Proteome changes in Aspergillus-infected human bronchial epithelial cells

Amreen Toor1,2, Luka Culibrk1,2, Gurpreet Singhera2, Kyung-Mee Moon1, Margo

Moore3, Anna Prudova1, Leonard Foster1, Delbert Dorscheid1,2,5 and Scott Tebbutt1,2,4,5

1University of British Columbia, Vancouver, Canada; 2 Centre for Heart Lung Innovation, St. Paul’s Hospital,

Vancouver, Canada; 3Department of Biological Sciences, Simon Fraser University, Burnaby, Canada; 4Prevention of Organ Failure (PROOF) Centre, Vancouver, Canada; 5Department of Medicine, Division of

Respiratory Medicine, UBC, Vancouver, Canada

Background Aspergillus fumigatus (A. fumigatus) is a ubiquitous filamentous fungus that disseminates in the air through the release of spores. Upon inhalation and binding/internalization by airway epithelial cells, spores may cause an infection in immunocompromised individuals. Our aim was to identify key host proteins and pathways to provide insights into the molecular mechanisms of this dual-organism interaction. Hypothesis Exposure of bronchial epithelial cells to A. fumigatus spores affects host gene expression. Methods Differentiated air-liquid interface (ALI) cultures of primary human bronchial epithelial cells (HBECs) were incubated with and without A. fumigatus spores for 6 hours (n=3). Proteins were extracted and analyzed by LC-MS/MS using stable-isotope labeling for quantification. MaxQuant software and LIMMA (R package) were used to identify differentially abundant proteins. Results Of the 2909 proteins identified, 1181 were quantified. Eight-three host proteins were differentially abundant in response to spore exposure (21 up-regulated and 62 down-regulated) (P value <0.05). Conclusion Shotgun quantitative proteomics identified candidate proteins and pathways to be validated in functional assays as potential mediators of A. fumigatus pathogenesis.

POSTER #34

Normoxic Regulation of Inducible Nitric Oxide Synthase expression by Hypoxia Inducible Factor in Human Cells

Martin Lee, Christine Wang, and Jonathan C. Choy

Department of Molecular Biology and Biochemistry, Simon Fraser University

Background: Inducible nitric oxide synthase (iNOS) synthesizes the bioactive gas NO, which has important inflammatory and immune regulatory properties. Hypoxia inducible factor (HIF) is a transcription factor essential for the cellular response to hypoxia as well as in the regulation of cell metabolism. While the regulation of iNOS expression by hypoxia has been demonstrated in murine models, the mechanism by which human iNOS gene expression may be regulated by HIF in inflammatory conditions is not understood. Here we demonstrate a role for HIF-1α in controlling cytokine-induced iNOS gene expression in normoxic conditions. Methods: iNOS expression was induced in the human epithelial cell line (A549) by stimulation with cytokines (CM; IL-1, IFNγ, and TNFα). The effect of inflammatory cytokines on HIF activity was examined by Western blotting for nuclear HIF-1α levels and through the use of HIF-response-element (HRE)-luciferase reporter assays. The effect of HIF on human iNOS expression was examined using a pharmacological HIF-1α inhibitor PX-478. Results: Cytokine stimulation of human cells did not affect HIF-1α expression levels at early time-points (2 h) but significantly increased nuclear HIF-1α levels. Cytokines induced the expression of iNOS mRNA and protein as well as the synthesis of NO. iNOS protein expression and NO levels were detectable beginning at 3 hours post-stimulation. Inhibition of HIF-1α expression by pharmacological inhibition markedly decreased cytokine-induced iNOS promoter activation as well as iNOS mRNA and protein expression. Conclusion: Induction of human iNOS gene expression by cytokines is dependent on HIF activity.

POSTER #35

Cloning rabbit monoclonal antibodies from single B cells and design of DNA constructs supporting their expression

Akesh Sinha1, Naveed Gulzar1 and Jamie K. Scott1, 2

1Department of Biochemistry and Molecular Biology, 2Faculty of Health Sciences, Simon Fraser University,

8888 University Drive, Burnaby, BC, Canada V5A 1S6

The spread of HIV epidemic is a global public health concern. There is major interest in developing HIV vaccines that could elicit potent broadly neutralizing antibodies (NAbs) against HIV. In particular, we are interested in using the conserved epitopes in the membrane-proximal external region of HIV gp41 subunit (MPER) of the envelope glycoprotein for vaccination. To this end, we have designed various DNA vaccine constructs that express the MPER tethered, via a transmembrane domain to the cell-surface and that may present it in a native-like form. In order to characterize Abs elicited by our MPER vaccines, it is best to employ single-cell based monoclonal Ab (mAb) cloning techniques. As a starting point, we have optimized a generalized method for specifically amplifying the variable region of rabbit antibody light and heavy chains from single isolated B cells. Furthermore, we have also developed a strategy for cloning the antibody variable regions in a suitable DNA background to generate rabbit-human chimeric mAbs, whose variable (V) domains are derived from the mRNAs from rabbit B cells, and whose constant (C) domains are derived from human IgG1, kappa and lambda heavy and light chains, respectively. We are currently working towards optimization of similar generic strategies to clone mAbs from humans, rhesus macaques and mice. In parallel, we are working towards designing strategies for easy purification of monoclonal antibodies expressed in tissue culture. Such strategies will allow us to produce and analyze the Abs expressed by antigen-specific B cells elicited from our HIV vaccine efforts. Finally, this study provides a general workflow for cloning antigen-specific mAbs from rabbits.

POSTER #36

Human T cell isolation, activation and expansion using EasySep™ and ImmunoCult™

Venus Lau, Jessie Yu, Andy I. Kokaji, Victoria Ng, Steven M. Woodside, Allen C. Eaves, and Terry E. Thomas

STEMCELL Technologies Inc., Vancouver BC, Canada

Cancer immunotherapy using chimeric antigen receptor (CAR) T cells is a rapidly progressing field and optimized protocols for the scalable manufacture of these cells are essential. We have developed novel products for T cell isolation, activation and expansion, and aim to make these products available as cGMP-grade reagents for use in cell therapy manufacturing. With our EasySep™ Release immunomagnetic cell separation system, highly purified, particle-free T cells can be obtained directly from unprocessed leukapheresis samples or cryopreserved PBMCs. Our soluble ImmunoCult™ Human T Cell Activators facilitate gentle activation of T cells, and together with our serum- and xeno-free ImmunoCult™-XF T cell expansion media, support the robust expansion of T cells. To better characterize our reagents, human T cells were activated and expanded in standard static cultureware, 24-well G-Rex gas permeable cell culture plates and the GE Xuri® W25 Cell Expansion System. Upon activation, human T cells showed transient upregulation of surface activation markers including CD25, PD-1 and 4-1BB and can be efficiently transduced with lentiviral vectors. We have found that the feeding strategy during the culture process is one of the most critical factors impacting total T cell expansion. With optimized culture protocols, up to 600-fold expansion of human T cells with greater than 85% cell viability was achieved over 10-14 days in all culture systems tested. Taken together, we have developed a streamlined scalable workflow solution for the isolation, activation and expansion of human T cells in serum-free culture conditions to enable the advancement of these breakthrough CAR T cell therapies.

POSTER #37

Arp 2/3-dependent spatial organization of the B cell receptor (BCR) impacts immune synapse formation, BCR signaling output,

and B cell activation

Madison Bolger-Munro1, Kate Choi1, Josh Scurll2, May Dang-Lawson1, Libin Abraham1, Lydia Yeo1, Duke Sheen1, Henry Lu1, Daniel Coombs2, Michael Gold1

1Department of Microbiology & Immunology and 2Department of Mathematics, University of British Columbia,

Vancouver, BC Canada The immune synapse is a contact-dependent cellular communication platform that is important for activating, amplifying and executing immune responses to pathogens. A B cell immune synapse is formed when antigens displayed on the surface of an antigen-presenting cell (APC) are recognized by the B cell receptor (BCR). This triggers dynamic, multiscale reorganization of the BCR, other membrane proteins, and signal transduction machinery, creating distinct molecular patterns that are optimized for cellular responses. Initially, BCRs are gathered into microclusters dispersed throughout the B cell:APC contact site. Microclusters then coalesce into a centralized domain at the center of the contact site. Although the clustering of BCRs into microclusters and subsequent microsignalosome formation increases signaling efficiency, how the formation of these dynamic spatial patterns of BCRs and signaling enzymes contributes to the interpretation of extracellular information (recognition of membrane-associated antigens) and subsequent cellular responses is not fully understood. The molecular mechanisms that control BCR movement and organization during immune synapse formation are therefore critical determinants of B cell activation. By imaging the B cell:APC contact site in real time, we tested the hypothesis that the evolution of BCR patterning from distinct and dispersed microclusters into the centralized immune synapse modulates not only the quantity, but also the quality of the B cell response to membrane-associated antigen. Our data show that the coalescence of BCR microclusters into the immune synapse, but not the formation of BCR microclusters, requires the dynamics of branched actin networks nucleated by the Actin Related Protein 2/3 Complex (Arp2/3). By blocking this machinery with Arp3 siRNA or an Arp2/3 inhibitor, we assessed the impact of pattern development and evolution on BCR signaling and the initiation of B cell activation programs. We found that BCR pattern evolution is important for amplifying initial BCR signaling in response to APC-bound antigens but not non-polarized soluble antigens. Additionally, the formation of the immune synapse is important for BCR-induced transcriptional responses and for the upregulation of B cell activation markers such as CD69 and CD86. This suggests that the dynamic patterning of BCRs is controlled by branched actin networks and can encode information that is pertinent to cellular decision-making, in this case B cell activation.

POSTER #38

Envelope-based Vaccines for the Concentrated HIV Epidemic in Saskatchewan

Manrubby Dhillon1, Naveed Gulzar1, Zabrina Brumme1, Andrew Ward2, Paul Levett3,

Rogier Sanders4, Jeff Joy5, Jamie K. Scott1

1Simon Fraser University, Burnaby, BC; 2The Scripps Research Institute, La Jolla, CA; 3Saskatchewan Disease Control Laboratories, Regina, SK; 4University of Amsterdam, Netherlands; BC Centre for Excellence

in HIV/AIDS, Vancouver, BC Designing an effective vaccine against HIV is a challenge due to this virus’s high genetic diversity. While the viruses circulating in most populations are highly diverse, the viruses in concentrated HIV epidemics have limited genetic diversity; this is the case for the HIV epidemic in Saskatchewan (SK). Phylogenetic analysis of polymerase gene (pol) sequence data in SK indicates multiple “clusters” of closely related viruses that have persisted and expanded over the past 12 years. Our objective is to develop and test envelope (Env)-based vaccine immunogens derived from viruses from the major pol clusters in SK. We have devised a strategy for cloning full-length Env genes (envs) from patient-derived plasmas. Two full-length Envs were functional, supporting infectious pseudoviruses (PsVs). These PsVs, were of higher titre and infectivity than a standard clade-B primary Env, SF162.LS, indicating SK Envs may be highly adapted to replicative fitness. Three Envs were engineered into trimeric gp140 vaccine candidates (SOSIPs). Cryo-EM analysis of purified trimers from one SOSIP showed >95% had a stable, “closed” trimeric form, similar to native trimer structures on viral particles. The SOSIPs were further analyzed in ELISA against monoclonal, HIV-neutralizing (N) antibodies (Abs) that bind “quaternary” (Qt) epitopes on Env trimers; such Abs indicate sites of “native-like” folding on Env. NAb binding profiles for each SOSIP did not fully reflect the neutralization sensitivity of the corresponding Envs borne by PsVs. For some NAbs, ELISA binding to SOSIP was low, yet neutralization activity against the corresponding PsV was strong, indicating some Qt epitopes on a native, infectious Env were altered after conversion into a SOSIP. Thus, while SOSIPs produced from SK envs may adopt a native-like overall Qt structure, some Qt epitopes may be less well defined. Whether SOSIP-immunogens will elicit cross-NAbs against closely related Envs in a pol cluster, and thus, provide protective immunity against them, will depend on the degree of Env variability within each cluster. If this vaccine strategy reduced HIV infection rates in SK, it would constitute the first proof of protection by vaccine-elicited NAbs.

POSTER #39

IL-7 is critical for CD8 T cell anti-viral effector responses during acute influenza/A infection

1Sheikh A.*, 1Plumb A.W., 1Seo J.H., & 1, 2Abraham N

1Department of Microbiology and Immunology, 2Department of Zoology, Life Sciences Institute, University of

British Columbia, Vancouver, B.C., Canada V6T 1Z3 Influenza/A virus infects airway epithelial cells and activates a network of immune cells. Following the innate responses, adaptive immune cells, particularly cytotoxic CD8 T cells, play a crucial role in controlling viral replication by killing infected cells. Proper activation, proliferation and function of these immune cells is necessary to clear infections efficiently. Interleukin-7 (IL-7), is a cytokine known for its importance in T cell development and survival. While the function of IL-7 in T cell survival is well characterized, it is unclear how IL-7 shapes T cell effector responses when a pathogen is encountered. To evaluate this, we used IL-7 receptor α mutant (IL-7Rα449F) mice, which have a tyrosine to phenylalanine substitution in the cytoplasmic domain of the IL-7 receptor α essential for signaling. Using this model, we have previously shown that IL-7 signaling is necessary for viral clearance in the lungs upon influenza/A infection. However, the mechanism by which IL-7 determines this remains unknown. We hypothesize that IL-7 is essential in generating unimpaired effector CD8 T cells during an acute influenza/A infection. Here, we show that IL-7Rα449F mice have fewer influenza-specific CD8 T cells in the lungs and draining mediastinal lymph nodes at days 5 and 7 post infection, as well as defective CD8 T cell degranulation as suggested by CD107a assays. Interestingly, IL-7Rα449F mice also accumulated lung CD11bhi dendritic cells (DCs), indicating either failed maturation and/or impaired migration to the draining lymph nodes, and thus, reduced CD8 T cell priming. These results suggest that IL-7 plays a critical role in the priming and effector functions of CD8 T cells during an influenza/A response.

This work was funded by the Canadian Institutes of Health Research, MOP-126060

POSTER #40

Increased T Follicular Helper Cell Infiltration in Lung Adenocarcinoma Tertiary Lymphoid Organs

Kevin W Ng, Erin A Marshall, Katey SS Enfield, Wan L Lam

Department of Integrative Oncology, BC Cancer Research Centre

T follicular helper cells (Tfh) are a CD4+ T cell subset that have been found to play crucial roles in the development of humoral immunity. In particular, their presence in the B cell-rich germinal centre of secondary and tertiary lymphoid tissue aids in B cell maturation through the promotion of somatic hypermutation, affinity maturation, and memory cell generation. Tfh cells have known roles in autoimmune disease and B cell malignancies; however, their role in many solid tumours, including those of the lung, remains less studied. We analyzed 83 paired tumour-normal lung adenocarcinomas from the BC Cancer Agency as well as 512 unpaired samples from The Cancer Genome Atlas (TCGA). Relative immune cell content was obtained from gene expression data using a linear support vector regression deconvolution approach (CIBERSORT). Spatial positioning of B and T cells within tumour tissue was examined through tumour microarrays. Clinical features, including patient survival, were analyzed using a Cox Proportional Hazard model. T follicular helper cells are increased in tumour tissue, accompanied by global upregulation of Tfh markers PDL1 and CXCR5 in both cohorts. Histological analysis revealed localization of Tfh cells within tertiary lymphoid organs, consistent with their role in providing B cell help. Importantly, Tfh recruitment appears to be a function of neoantigen exposure rather than simply chronic inflammation. T follicular helper cells are required for B cell maturation and subsequent antibody responses. As such, it is not surprising that Tfh infiltration in tumour-resident ectopic lymphoid structures correlates with patient survival in various cancer types. Given the importance of tumour-specific antibody responses in natural and therapeutic immunity, further investigation of Tfhs may show great therapeutic and prognostic utility.

POSTER #41

Investigating the Functional Evolution of HIV Nef Protein within Host

Hanwei Sudderuddin1, N. Kinloch1, S. Chandrarathna1, P.R. Harrigan2, M. Brockman1,2, Z. Brumme1,2

1. Faculty of Health Sciences, Simon Fraser University; 2. BC Centre for Excellence in HIV/AIDS

Over 30 years have elapsed since HIV-1 was first recognized as the causative agent of AIDS. While advances in antiretroviral therapies have transformed HIV into a chronic manageable condition, a vaccine is ultimately needed to curb the global pandemic - but HIV's extensive capacity for mutation and genetic diversification remains a major impediment to this goal. Studies in human hosts with HIV-1 have identified the Nef protein to be a major determinant of AIDS pathogenesis. The nef gene, which is well-conserved in primate lentiviruses, exhibits pleiotropic properties and is found at the 3' end of the HIV genome. Although only ~ 27kDa in size, Nef is known to perform a multitude of immunomodulatory functions that enhance viral replication, infectivity and immune evasion. For example, Nef’s abilities to downregulate CD4 and Human Leukocyte Antigen (HLA) class-I from the infected cell surface have been especially well characterized. Reduction of CD4 cell-surface molecules allows release of budding HIV virions from infected T cells and this appears to promote viral infectivity. The down-modulation of HLA-I by Nef has been implicated in protecting HIV infected cells from NK cell cytotoxicity. A key feature of HIV is its rapid rate of evolution within an infected host. For instance, following productive infection with a single founder virus (on average), within-host genetic diversity, and divergence from the founder, expands substantially over time. Given Nef’s variety of functions and the high level of HIV evolution observed during infection, it would be useful to assess if Nef function remains static or changes over time within an infected host. My undergraduate honors project will address this question by functionally characterizing longitudinally isolated Nef sequences from at least one patient over a follow-up period of 10 years during which ART was not administered. A longitudinal Nef plasma HIV RNA sequence dataset for the first study patient, comprising 113 unique sequences collected between 1996 and 2007, has already been generated by limiting-dilution RT-PCR followed by Sanger sequencing. As expected, the data indicate substantial within-host HIV evolution over time: of Nef's 207 codon positions in this patient, non-synonymous amino acid substitutions are observed at 37 (17.87%) sites; one sequence additionally harbored a single amino acid deletion. I am now in the process of selecting 50 unique Nef sequences sampled across the study period for functional assessment. The selected Nef sequences will be re-amplified, cloned into pSELECT-GFP, re-sequenced, and expressed in immortalized CD4+ T cells. Nef’s relative CD4 and HLA-I down-modulation abilities in the patient-derived Nef clones will be assessed using flow-cytometry assays. It is hoped this will achieve two outcomes: 1) illuminating the extent to which Nef-mediated CD4 and HLA-I downregulation functions change within-host during HIV infection, and 2) generating a paired genotype-phenotype dataset that will allow identification of sequence determinants of Nef functional activity.

POSTER #42

Investigating Granzyme B involvement in dermo-epidermal separation in blistering skin conditions

Russo V.1,2, Klein T.3,4, Carr N.J.5, Van Laeken N.5, Crawford R.I.6, Overall C.M.3,4,

Granville D.J.1,2

1 ICORD Research Centre, Vancouver, BC, Canada V5Z 1M9 2 Department of Pathology and Laboratory Medicine, UBC, Vancouver, BC, Canada V6T 2B5 3 Centre for Blood Research, UBC, Vancouver, BC

Canada V6T 1Z3 4 Department of Oral Biological and Medical Sciences, UBC, Vancouver, BC Canada V6T 1Z3 5 Department of Surgery, UBC, Vancouver, BC, Canada V5Z 1M9 6 Department of Pathology and

Laboratory Medicine UBC, Vancouver, BC, Canada 7 Department of Dermatology and Skin Science, Faculty of Medicine, UBC, Vancouver, BC, Canada

Introduction: Blistering skin disorders are a group of rare skin diseases involving blistering and erosions in the skin and/or mucous membranes. Although the etiology of these conditions can be autoimmune, genetic or related to hypersensitivity to certain drugs, they all involve epidermal detachment with consequent increased risk of infection and reduced quality of life. In healthy skin, epidermis and dermis are anchored together by the dermal-epidermal junction (DEJ), a specialized basement membrane pivotal for the integrity and function of the skin. Granzyme B (GzmB) is a serine protease secreted by immune cells that can cleave extracellular matrix proteins. Although elevated levels of GzmB have been found at the level of the DEJ in inflammatory and blistering skin conditions, the present study is the first to explore GzmB ability to cleave DEJ components, thus causing epidermal detachment. Materials and Methods: Presence of GzmB in skin obtained from patients affected by several skin blistering conditions was confirmed through IHC. In order to define a potential role of GzmB in mediating cleavage of important components of the DEJ, cleavage assay and TAILS analysis were performed on Collagen VII, α6β4 integrin and Nidogen-2. Finally, samples of healthy skin were exposed to GzmB to investigate in vitro DEJ cleavage through H&E and IHC staining. Results: Significant presence of GzmB could be observed in the DEJ of diseased human skin, and epidermal detachment was observed in healthy skin upon exposure to GzmB. Western Blot analysis showed GzmB-mediated cleavage of Collagen VII, α6β4 integrin and Nidogen-2, which could be prevented by GzmB inhibitors. Furthermore, mass spectrometry analysis revealed that these DEJ proteins are cleaved in a region crucial for their anchoring function. Conclusion: Overall our data suggest that GzmB could be responsible for epidermal detachment observed in blistering skin conditions and thus represents a potential target for their treatment.

POSTER #43

iRECEPTOR: Bioinformatics platform for storing and sharing next-generation sequencing data from immune repetoires

Felix Breden1, Nishanth Marthandan1,2, Bojan Zimonja1, Jerome Jaglale1, Nicole

Knoetze1, Emily Barr1, Richard Bruskiewich1, Yang Zhou1, Frances Breden1, Jamie Scott1,2,3, and Brian Corrie4

1IRMACS Centre, 2Dept Molecular Biology & Biochemistry, and 3Faculty of Health Sciences, Simon Fraser

University, Burnaby, BC; 4Centre for eResearch, Auckland, New Zealand The adaptive immune system comprises the collection of B cells, plasma cells (AKA antibody-secreting cells), and T cells in a vertebrate organism. Adaptive-immune receptor repertoires (AIRRs) comprise the collection of B-cell receptors, antibodies, and T-cell receptors expressed in a collection of B cells, plasma cells and/or T cells. The diversity of the human AIRR is vast; ~1013 B-cell receptor/antibody sequences are possible, far outstripping the ~1011 cells that can fill the B- and T-cell nicheses in the human body. Next-generation has enabled the collection of AIRR data, which have become critical for development of vaccines and therapeutic-antibodies and other immunotherapies. A major caveat to AIRR NGS data is the technical difficulties in sharing and storing them; yet, only by analyzing data across studies, with associated metadata on patients, their treatments and clinical outcomes, will the potential of this revolution in immunotherapeutics be realized. The iReceptor Gateway currently under development will link AIRR databases among institutions, allowing them to be queried for sequences satisfying specific criteria. iReceptor implements a distributed data set model, such that researchers can store their data under their own IRB constraints, and release these data to other nodes in the iReceptor environment based on shared consent, reciprocal data transfer, and IP agreements. In order to address the challenges involved in storing and sharing these AIRR NGS data, a group of immunologists, bioinformaticists, ethicists, lawyers and others have formed a consortium to develop protocols and best practices for producing, reporting and analyzing AIRR data. You are invited to join us at the next Adaptive Immune Receptor Repertoire Community Meeting, to be held December 2017 at the NIH Fishers Lane Facility in Rockville, MD. For additional information and/or to be added to the mailing list contact Felix Breden at: breden@sfu.ca.

POSTER #44

Antagonism of SERINC5 is impaired in Nef clones isolated from HIV-1 elite controllers

Steven W. Jin1, Nirmin Alsahafi2,3, Xiaomei T. Kuang1, Heinrich Göttlinger4, Andrés

Finzi2,3,5, Zabrina L. Brumme1, 6, Mark A. Brockman1, 6

1Simon Fraser University; 2CRCHUM; 3McGill University; 4University of Massachusetts, USA; 5Université de

Montréal; 6British Columbia Centre for Excellence in HIV/AIDS

Background: Nef is multifunctional accessory protein that is critical for viral pathogenesis. It was recently reported that Nef enhances virion infectivity by downregulating host restriction factor SERINC5 from the cell surface. However, no studies have evaluated the impact of naturally occurring Nef polymorphisms on this activity, particularly in the context of non-pathogenic clinical outcomes. Here, we examined the ability of Nef alleles from HIV-1 elite controllers (EC), patients who spontaneously suppress plasma viremia without antiretroviral therapy, to antagonize SERINC5. Methods: Nef alleles from 45 EC and 46 chronic progressors (CP) were amplified from plasma viral RNA and cloned. Nef-mediated downregulation of SERINC5 from the cell surface was assessed by flow cytometry and results were normalized to a WT Nef strain. Polymorphisms associated with differential function were identified using statistical analyses of linked Nef genotype-phenotype data. Infectivity was measured by exposing TZM-bl reporter cells to HIV-1 particles (5ng p24) Results: Nef clones derived from EC displayed lower ability to antagonize SERINC5 (median 80 [IQR 38-95]%) compared to clones from CP (96 [IQR 75-100]%) (p=0.0005). A genotype-phenotype analysis of these 91 Nef alleles identified an association between H116N and reduced downregulation function (H: 95% vs. N: 81%; p=0.01). Moreover, an HIV-1 strain encoding the Nef H116N mutation displayed 36% infectivity relative to WT virus. Notably, H116N is a CTL escape mutation selected by patients that carry the protective HLA-B*57 gene, which is associated with slower disease progression and is enriched among EC. Conclusions: Our results demonstrate that EC Nef clones are attenuated for their ability to antagonize SERINC5. Natural sequence variation in Nef, including polymorphisms selected to evade CTL pressure, could have significant impact on this function. Differences in Nef’s ability to downregulate SERINC5 may contribute to improved clinical outcomes in some EC.

POSTER #45 Functional validation of sequence-based analyses of antigen specific T-

cell receptor repertoires

G Anmole1, S Jin1, XT Kuang1, S Mujib2, J Wang1, MA Ostrowski2, MA Brockman1,3

1Simon Fraser University, Burnaby BC; 2University of Toronto, Toronto ON; 3BC Center for Excellence in HIV/AIDS, Vancouver, BC.

Background: TCR repertoire diversity determines the breadth and specificity of an individual's CTL response against HIV infection. A better understanding of features associated with highly active antiviral TCR restricted against HLA-A*02 Gag FK10 may inform TCR based immunotherapeutic strategies to target the latent HIV-1 reservoir. The high prevalence of HLA-A*02 in North American population and the immunodominance of FK10 epitope make this combination an attractive TCR immunotherapy target. Methods: Tetramer+ CD8 T-cells against Gag FS8 (FLGKIWPS) were isolated by FACS from HLA-A*02+ HIV non-progressors. Paired TCR alpha and beta CDR3 sequences were determined using single-cell RT-PCR. Full-length TCR genes were reconstructed and transfected into Jurkat cells along with CD8 alpha and NFAT-driven luciferase reporter. TCR+ Jurkat “effector” cells were co-cultured with A*02+ “target” cells pulsed with FK10 peptide or variants and TCR-mediated signaling was quantified by luminescence. Results: A dominant TCR clonotype reactive against A2-FK10 was observed in two unrelated individuals. In one case, clone 1A9 was observed at ~60% frequency in tetramer-sorted cells. In the second case, clone 4A4 was observed at 100% frequency. 1A9 and 4A4 encoded identical alpha and beta Variable genes (TRAV12-1, TRBV7-2). Furthermore, their alpha CDR3 sequences differed by 1 amino acid, and their beta CDR3 sequence differed by 4 amino acids. 1A9 and 4A4 showed similar antigen sensitivity to FS8 (EC50: 0.29 ng/µL and 0.23 ng/µL, respectively) and FK10 (EC50: 2.1 ng/µL and 0.97 ng/µL) in a TCR signaling assay. Analysis of FS8 variants indicated that positions 4, 6 and 7 were critical for signaling by both TCR, and a natural polymorphism at peptide position 4 (Gag K436R) similarly abrogated their activity. Conclusions: We have established a system to characterize the sequence and function of antigen specific TCR repertoires and utilized it to identify a novel public TCR clonotype against A2-FK10 with nearly identical sequences, signaling capacities and peptide cross-reactivity profiles. Our results indicate that the T cell response elicited towards this HIV epitope may be highly restricted, but effective once generated. These results, combined with future studies, may contribute to the design of TCR based strategies for HIV eradication.

POSTER #46

Tuft cells and their role in intestinal inflammation

Jean Sauvé1, Roger Jen1, Keith McLarren1, Hayley Brugger1, Eyler Ngoh1, Theodore Steiner2 and Laura Sly1

1 Department of Paediatrics, University of British Columbia and BC Children’s Hospital Research Institute 2 Department of Microbiology & Immunology, University of British Columbia, Vancouver, BC, Canada

Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, is characterized by intestinal inflammation. Intestinal epithelial cells play a critical role in mucosal homeostasis and dysregulation of pro-inflammatory epithelial cell function could lead to the intestinal inflammation that characterizes IBD. However, we do not know the events that initiate inflammation or the cell types involved. One type of cell that may play a role is the tuft cell. Tuft cells are the only epithelial cells in the uninflamed intestine that express COX-1 and COX-2, the rate-limiting enzymes required for production of prostaglandins, like pro-inflammatory PGE2. In our research investigating the lipid phosphatase SHIP, we discovered that tuft cells express SHIP. SHIP deficiency leads to increased PI3-kinase activity in cells resulting in increased cell proliferation, reduced apoptosis, and increased cell activation. SHIP expression is currently believed to be restricted to hematopoietic cells. However, using bone marrow transplantation, we found that tuft cells were not radiosensitive, suggesting that they are not bone-marrow derived and are not hematopoietic in origin.

In addition, SHIP deficient mice develop spontaneous Crohn’s disease-like intestinal inflammation. Onset of inflammation coincides with the developmental appearance of tuft cells. In wild type mice, tuft cells are found in the lung and ileum, both locations where SHIP deficient mice develop spontaneous inflammation, and we found that tuft cell numbers were increased 6-fold in the inflamed ileum of SHIP deficient mice. Based on this, we hypothesized that SHIP deficient tuft cells may initiate inflammation in the SHIP deficient mouse. We found that SHIP deficient mice had more COX-1 positive cells in the ileum and more COX activity, PGD2 and PGE2 in full thickness ileal tissue homogenates, compared to their wild type littermates. Finally, prophylactic inhibition of COX activity with piroxicam, reduced the development of intestinal inflammation in SHIP deficient mice whereas therapeutic treatment had no effect. This suggests that tuft cells may be critical in the initiation of spontaneous intestinal inflammation in SHIP deficient mice.

POSTER #47

Exploring the Role of Cellular Reprogramming During Sepsis

Beverlie Baquir, Erin Gill, Amy Lee, Robert Hancock

Centre for Microbial Diseases and Immunity Research, Department of Microbiology and Immunology, University of British Columbia

Sepsis is an assemblage of symptoms resulting in a dysregulated host immune response to severe infection and causes over 5 million deaths annually. It has been reported that early identification and diagnosis has the potential to improve patient outcomes and survival. Since sepsis pathogenesis is poorly understood, there is a great need to define and identify the factors that are at play, especially during early sepsis. We have performed a meta-analysis of the transcriptional responses from more than 600 sepsis patients and have discovered a proclivity towards a cellular reprogramming (CR) gene expression signature. Very early sepsis patients showed strong gene expression signatures reflecting CR and immunosuppression, indicating a potential new major immune perturbation during disease progression. CR which includes endotoxin tolerance (ETT), is marked by stunted responses of innate immune cells and this reprogrammed state is predictive of the development of subsequently-diagnosed severe sepsis and organ failure in sepsis. Thus, while a mixed pro- and anti-inflammatory state in early sepsis has been suggested, it is worth noting that CR is a form of cellular amnesia rather than an anti-inflammatory state per se. With that in mind, the goal is to uncover the mechanisms underlying cellular reprogramming. Monocytes/macrophages possess the plasticity to change functional states under the appropriate stimuli. It was previously shown that reprogrammed macrophages have a hampered ability to produce pro-inflammatory cytokines in response to microbial signatures such as lipopolysaccharide and in fact, adopt a M2-like differentiation state. Therefore I hypothesize that monocytes/macrophages are the major cell population driving cellular reprogramming in sepsis patients and that in vitro macrophage cells derived from human induced pluripotent stem (iPS) cells that are reprogrammed can be used to mimic and study cells expressing the cellular reprogramming gene expression pattern associated with early sepsis.

POSTER #48

An algorithm for inferring immunoglobulin variable-region germline genes

Nishanth Marthandan1, Jeffrey B. Joy4, Felix J. Breden2 and Jamie K. Scott1,3

1 Department of Molecular Biology and Biochemistry, Simon Fraser University, 2 Department of Biological

Sciences, Simon Fraser University, 3 Faculty of Health Sciences, Simon Fraser University, 4 Centre for Excellence in HIV/AIDS, St. Paul’s Hospital; Dept. of Medicine, Faculty of Medicine, University of British

Columbia

B-cell clonal expansions are a major component of normal immune responses to infectious pathogens and vaccines. However, aberrant expansions of B cells play a role in the development of B-cell leukemias, and autoimmune diseases. Understanding clonal expansions should inform vaccine design and treatment strategies. In studying clonal expansions, it is critical to identify clonal lineages, which comprise B-cell clones that have accrued somatic hypermutations (SHMs) in the genes encoding immunoglobulins (Igs) and B-cell receptors (BcRs). SHMs arise during B-cell responses to infection and vaccination, and in inflammatory and autoimmune states. SHMs are identified by comparing the BcR gene-sequence with the germline genes (GLGs) that initially encoded those expressed sequences. Public databases, such as the IMGT→, comprise incomplete sets of known GLGs, which can to lead to incorrect determination of SHMs, and therefore, misidentification of clonal lineages. The main purpose of my thesis work is to develop and validate a novel computational algorithm for inferring GLGs. My algorithm analyzes both “simulated” and “real” next-generation sequencing (NGS) data encoding Ig and BcR repertoires to infer GLGs based on their usage in multiple, different, recombined Ig and BcR sequences. The algorithm involves combining one of two strategies for identifying IGHV gene segments within a set of Ig or BcR sequences, with one of two strategies for inferring GLG sequences. Thus, four different algorithm-modules are validated using BcR repertoire datasets simulated from the C57BL/6 GLG repertoire, which is completely known. Each algorithm will be optimized by finding the optimal parameters for the specific characteristics of several NGS BcR repertoires. This is accomplished by comparing the confidence scores assigned to the inferred germline genes, by each algorithm, and comparing the overall measures of the algorithms’ performance on the simulated datasets. Preliminary results of my work in the development and validation of the algorithms are presented.

POSTER #49 Our favourite FLVR: Probing the asthma-associated immunomodulatory

effects of the gut microbiota

Rozlyn C.T. Boutin1, Jorge A. Peña-Diaz1, Sarah E. Woodward1, B. Brett Finlay1,2,3

1Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC, Canada

2Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada 3Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada

Asthma is an incurable inflammatory disease of the airways that typically presents in childhood and affects over 300 million people worldwide. An increasingly large body of evidence suggests that appropriate colonization of the infant gastrointestinal tract by symbiotic microbes is essential to the development of a healthy human immune system, and that abnormal or “dysbiotic” gut microbiota communities in early life may contribute to the etiology of asthma. However, the exact mechanisms mediating the asthma-protective or -predictive effects of specific gut microbes on the host immune system remain elusive. Our lab has identified four bacterial genera that are reduced in abundance in the feces of 3-month old children at high risk of developing asthma as compared to healthy children from the Canadian Infant Longitudinal Development (CHILD) study. These genera include Faecalibacterium sp., Lachnospira sp., Veillonella sp., and Rothia sp. (FLVR). We have also determined that these changes in abundance do not persist at one year of age, indicating a role of FLVR in host immune development. Moreover, we used a murine model of asthma and human-to-mouse fecal transplants using stool from a 3-month old infant who later developed asthma spiked with representative FLVR species to demonstrate a causal relationship between FLVR deficiency and asthma exacerbation. Based on preliminary findings showing reductions in colonic regulatory T cells (Tregs) and increased circulating IgE levels in asthma-susceptible antibiotic-treated mice, we hypothesize that FLVR species promote tolerance and prevent allergic asthma responses by promoting the induction of Tregs and inhibiting allergic Th2-associated IgE antibody production and colonic eosinophil accumulation. Here we present preliminary data and our plan to further characterize the effects of FLVR on immune function in early life. Investigations into host-microbiota interactions will allow us to determine which of the specific cell types associated with allergic asthma are influenced by FLVR species and thereby inform future work aiming to develop novel therapeutic tools capable of preventing or reducing the burden of childhood asthma.

POSTER #50

The Effect of Diet on the Microbiome Composition and Incidence of NNK-induced Lung Cancer

Ingrid Elisia, Mariah Hay, Michael Li, Brandon Cho, Elyse Hofs, Vivian Lam, Hilary

Leung, William Mohn, Gerald Krystal

The Terry Fox Laboratory, British Columbia Cancer Agency We investigated the efficacy of low-carbohydrate (CHO) diets in preventing 4-(N-methyl-N-nitrosamino)-1-(3- pyridyl)-1-butanone (NNK) - a tobacco-specific carcinogen -induced lung cancer in female A/J mice. In addition, we evaluated the role of different types of CHO (easily digestible vs resistant), protein (casein vs. soy) and fat (fish vs. coconut vs. a mixture of oils) in modulating lung nodule formation in these mice. To keep the chows isocaloric, the low CHO diets had elevated levels of fat and protein. The lowering of easily digestible CHO significantly reduced constitutive blood glucose levels and lung nodule formation in the mice. Interestingly, diets low in easily digestible starch, high in fish oil and soy protein were the most effective at preventing the formation of NNK-induced lung nodules. Changes in fecal microbiome composition were also associated with the diets. Changing protein, CHO and fat type in the diets all resulted in differences in the fecal microbiome composition of the NNK-injected mice. As an example, we found a reduced abundance of the Streptococacceae and Clostridiaceae families in mice with low lung nodule numbers. It is thus possible that the diets reduced lung nodule formation, at least in part, via alterations in the microbiomes of the mice.

POSTER #51

Microbiome-driven allergic lung inflammation is ameliorated by short chain fatty acids

Alissa Cait1, Michael R. Hughes2, Frann Antignano2, Jessica Cait2, Pedro A. Dimitriu1,

Kendra R. Maas1, Lisa A. Reynolds6, Lina Hacker2, Julia Mohr2, B. Brett Finlay1, 6,7 Colby Zaph4, 5, Kelly M. McNagny2, 3, William W. Mohn1

1Department of Microbiology and Immunology, LSI, UBC 2The Biomedical Research Centre, UBC 3Department of Medical Genetics, UBC 4Department of Pathology and Laboratory Medicine, UBC

5Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia 6Michael Smith Laboratories, UBC 7Department of Biochemistry and Molecular

Biology, UBC

The mammalian gastrointestinal tract harbors a dense community of microorganisms that are critical for our health. The microbiota contains vast metabolic potential and the metabolites produced by these microbes modulate immune cells directly. Short chain fatty acids (SCFAs) are small carboxylic acids produced in the colon by bacterial fermentation, and their levels are diminished in germ free and antibiotic treated mice. Recently, there has been interest in uncovering the role of SCFAs in regulating the immune system. We used the antibiotic vancomycin as a tool to deplete the SCFA–producing component of the microbiota. We found that mice treated with vancomycin have an altered microbiome and metabolite profile, exhibit exacerbated Th2 responses, and are more susceptible to allergic lung inflammation. We show that dietary supplementation with short chain fatty acids (SCFA) ameliorates this enhanced asthma susceptibility by modulating the activity of T cells and dendritic cells (DCs). Dysbiotic mice treated with SCFA have fewer IL-4-producing CD4+ T cells and decreased levels of circulating IgE. In addition, DCs exposed to SCFA activate T cells less robustly, are less motile in response to CCL19 in vitro and exhibit a dampened ability to transport inhaled allergens to lung draining nodes. Our data thus demonstrate that gut dysbiosis can enhance allergic lung inflammation through both T cell and DC-dependent mechanisms. By uncovering how SCFAs influence immune function we hope to gain greater insight into the complex regulatory networks linking the microbiota to chronic diseases, such as asthma.

POSTER #52

The role of the Innate Immune Response Regulatory Gene ABCF1 in Mammalian Embryogenesis and Development

Hitesh Arora1,2, Sara M. Wilcox1,2, Lonna Munro1, Franz Fenninger 1,2, Laura A. Johnson1,4, Pamela Hoodless5,7,8 and Wilfred A. Jefferies1-7

1The Michael Smith Laboratories, 2Department of Microbiology & Immunology, 3Department of Medical Genetics, 4Department of Zoology, 5Centre for Blood Research, 6Djavad Mowafaghian Centre for Brain

Health, 7Department of Developmental and Cell Biology, 8Terry Fox Laboratory, BC Cancer Agency, The University of British Columbia, Vancouver, Canada.

ABCF1 is an ABC transporter family protein that has been shown to regulate innate immune response and is a risk gene for autoimmune pancreatitis and arthritis. Unlike other members of ABC transporter family, ABCF1 lacks trans-membrane domains and is thought to function in translation initiation through an interaction with eukaryotic translation initiation factor 2 (eIF2). To study ABCF1 in development and disease, we used a single gene trap insertion in the Abcf1 gene in murine embryonic stem cells (ES cells) that allowed lineage tracing of the endogenous Abcf1 promoter by following the expression of a β-galactosidase reporter gene. From the ES cells, heterozygous mice (Abcf1+/-) were produced. No live born Abcf1-/- progeny were ever generated, and the lethality was not mouse strain-specific. Thus, we have determined that Abcf1 is an essential gene in development. Abcf1-/- mice were found to be embryonic lethal at 3.5 days post coitum (dpc), while Abcf1+/- mice appeared developmentally normal. Abcf1+/- mice were fertile and showed no significant differences in their anatomy when compared with their wild type littermates. The Abcf1 promoter was found to be active in all organs in adult mice, with varying levels in different cell types. Furthermore, we observed high promoter activity in the blastocysts and embryos. Overall, Abcf1 expression in embryos is required for development and its expression in adults was highly correlated with actively proliferating and differentiating cell types.

POSTER #53

Altered Receptor Mobility & Spatial Organization in Marginal Zone B cells

Abraham L1, Tafteh R2, Lu HY1, Scurll J3, Chappell R3, Sung KW3, Chou KC2,

Coombs D3, Gold MR1

1Department of Microbiology & Immunology, 2Department of Chemistry, 3Department of Mathematics, University of British Columbia, Vancouver, Canada

B cells integrate signals from multiple activating and inhibitory receptors in a highly regulated spatiotemporal manner to regulate B cell receptor (BCR) signaling and B cell activation. Marginal Zone (MZ) B cells are unique subset of B cells that exist in a partially activated ‘primed’ state, allowing them to rapidly respond to small amounts of antigens. The molecular basis for this priming is not understood. We propose that the priming of MZ B cells reflects altered lateral mobility and nanoscale organization of the BCR and other cell surface proteins, as compared to naïve circulating follicular (FO) B cells. To quantify receptor mobility and spatial organization, we used high-speed single particle tracking and multi-color super-resolution microscopy, respectively. We found that IgM, but not IgD BCRs, in MZ B cells have (i) higher lateral mobility, (ii) larger confinement zone, and (iii) higher slowàfast state transition rates, when compared to FO B cells. Using a novel, unsupervised, hierarchical clustering algorithm (STORMgraph), we quantified nanocluster area and density. We found that both IgM and IgD BCR isotypes exist as larger nanoclusters on the surface of MZ B cells, when compared to FO B cells. Although both BCR isotypes exist in discrete and heterogeneous nanoscale protein islands in B cells, signaling active BCRs predominantly overlapped with IgM containing nanoclusters, when compared to IgD. The overlapping of IgM BCR with ‘signaling hub’ protein islands (such as CD19 co-receptor) in MZ B cells may result in greater antigen-independent tonic signaling from the BCR, contributing to the partially-activated ‘primed’ state of MZ B cells.

POSTER #54

Alveolar macrophage lipid homeostasis is disrupted by CD44 deficiency

Yifei Dong, Arif A. Arif, Manisha Dosanjh, and Pauline Johnson

Department of Microbiology and Immunology, University of British Columbia

Alveolar macrophages maintain lung homeostasis by performing important roles in immunosurveillance and pulmonary surfactant catabolism. Patients and mouse models with defective alveolar macrophage development or depleted alveolar macrophage numbers develop pulmonary alveolar proteinosis due to excess accumulation of pulmonary surfactant. In addition, lung inflammation leads to the oxidation of surfactant lipids which are toxic to macrophages. Increased uptake of oxidized lipids can lead to their transformation into foam cells and excessive TGF-β production, which drives lung fibrosis. Thus, it is important to gain a better understanding of how lipid uptake and metabolism is regulated in alveolar macrophages and how these impact the resolution of lung inflammation and fibrosis. Alveolar macrophages express a high level of CD44 that helps anchor a protective hyaluronan coat around these cells and promoted their survival. This coat was absent in CD44-/- alveolar macrophages and as a result, they were present in lower numbers and were more susceptible to cell death in the CD44-/- lung. CD44-/- mice also had an increased concentration of the major component of the pulmonary surfactant, phosphatidylcholine, suggesting an insufficient number of macrophages present to catabolize surfactant lipids. CD44-/- alveolar macrophages showed elevated surface expression of CD36, a scavenger receptor capable of taking up fatty acids and oxidized phospholipids, and accumulated greater amounts of neutral and oxidized lipids compared to normal alveolar macrophages. This increased the susceptibility of CD44-/- alveolar macrophages to oxidized phospholipid toxicity. Together, these data show alveolar macrophage survival and lipid homeostasis is regulated by CD44 and hyaluronan and that defects in these processes may lead to reduced immunosurveillance, dysregulated surfactant metabolism and increased inflammatory and fibrotic responses, which may have new clinical implications.

POSTER #55

The Effect of the Protein Tyrosine Phosphatase, CD45, on regulating erythroid development and suppression of immune responses

Yaein Amy Shim, Teresa Campbell and Pauline Johnson

Department of Microbiology and Immunology, UBC

The receptor protein tyrosine phosphatase, CD45, is expressed on all hematopoietic nucleated cells and is lost as red blood cells mature and lose their nuclei. It dephosphorylates Src family kinases such as Lyn, thus regulating antigen and cytokine derived signaling pathways in immune cells. CD45 is present on erythroid progenitor cells and has been implicated in the negative regulation of erythropoietin (Epo) signaling in bone marrow erythroid progenitors. More recently, Ter119+CD71+ red blood cell progenitors have been implicated in immunosuppression in neonates. In this study, I wanted to understand how CD45 regulates erythroid development and its impact on the suppressive progenitor population. I observed that CD45 deficient mice retain a high level of Ter119+CD71+ erythroid progenitor cells in their spleen from neonatal to adult stage, whereas wild-type mice normally downregulate the erythroid progenitors by 3 weeks after birth. However, this did not lead to significant differences in mature RBC in the spleen or blood, suggesting that these progenitors might be short lived. Indeed, preliminary data showed that these cells express reduced levels of Bcl-2 but not Bcl-xL. Since CD45-/- mice had increased number of Ter119+ CD71+ erythroid progenitor, equivalent to those present in the neonates, I determined whether these cells were also capable of immunosuppressing dendritic cell activation. Incubation of LPS activated bone marrow derived dendritic cells (BMDC) with neonatal or adult wild-type or CD45-/- total red blood cell progenitors isolated from the spleen led to less TNFα and IL-12 production from BMDCs exposed to neonatal and CD45-/- cells. Further investigation revealed that the active cell population was the Ter119+ CD71+ population and that these cells were depleting the BMDC population. Furthermore, injection of this CD45-/- Ter119+ CD71+ population into mice was able to rescue the systemic wasting disease induced by inducing T cell colitis in Rag deficient mice. These observations suggest a novel role for CD45 in erythrocyte development prior to their enucleation and raises the possibility that the abnormal accumulation of specific erythroid progenitor cells may provide a more immunosuppressive systemic environment in CD45 deficient mice.

POSTER #56

Deciphering monocyte heterogeneity using mass cytometry

Maunish Barvalia*, Morgan E. Roberts* and Kenneth W. Harder*

* Infection, Inflammation and Immunity research group, Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC, Canada.

Monocytes are key immune cells which play a crucial role in maintaining tissue homeostasis, initiation of inflammation, and resolution of inflammatory responses. Monocytes belong to the mononuclear phagocyte system and murine monocytes express the integrin CD11b and colony stimulating factor-1 receptor (CSF-1R, also known as CD115). Currently murine monocytes are categorized into two major subsets based on the shared expression of CD11b and CD115 and their differential expression of the cell differentiation antigen Ly6C. Ly6Chi are designated as conventional monocytes, while Ly6Clow monocytes are known as non-conventional or patrolling monocytes.

Using mass cytometry by time of flight (CyTOF), a high throughput, multidimensional, single cell proteomic technique, we show here that monocytes are a highly heterogeneous population. CyTOF identified the two major subsets of murine monocytes in various tissues, but additionally revealed that both subsets are highly heterogeneous based on expression patterns of other cell surface proteins, and hence enables the identification of various subpopulations within the two major monocyte subsets. We also found that the two major monocyte subsets have very different signaling network profiles. In addition, various monocyte subpopulations also have subtle differences in their signaling network profiles. Loss of the src-family tyrosine kinase Lyn in mice leads to an expansion of the patrolling monocyte subset and impacts the expression of many cell surface markers and perturbs signalling patterns within particular monocyte subsets. Finally, we show that Lyn deficiency affects other immune cell populations leading to systemic changes in various immune system compartments.

POSTER #57 Investigation of in vitro Vpu function across major HIV-1 group M subtypes

Gisele Umviligihozo1, Kyle Cobarrubias1, Guinevere Lee2, Richard Harrigan2, Peter Hunt3,

Jeff Martin3, David Bangsberg4, 5, Zabrina Brumme 1, 2, Mark Brockman1,2

1. Faculty of Health Sciences, Simon Fraser University, Burnaby, BC, Canada 2. BC Centre for Excellence in

HIV/AIDS, Vancouver, BC, Canada 3. University of California, San Francisco, California, USA 4. Oregon Health Science University, Corvallis, Oregon, USA 5. Mbarara University of Science and Technology,

Mbarara, Uganda Background: The HIV-1 accessory protein Vpu is a multifunctional ~16kDa transmembrane protein that enhances viral replication and facilitates immune evasion. Two of Vpu's best-characterized functions are downregulation of the HIV entry receptor CD4 and the antiviral protein tetherin from the infected cell surface. Modulation of these molecules helps HIV-infected cells evade detection by innate and adaptive immune responses, and also enhances viral egress. A notable feature of HIV-1 is its extensive global genetic diversity: isolates of the HIV-1 group M "pandemic" strain can be classified into nine subtypes and recombinant forms emerge continually. Moreover, Vpu represents one of HIV-1's most genetically diverse regions. It is thus conceivable that genetically diverse Vpu isolates may exhibit differences in their ability to downregulate CD4 and Tetherin, which could in turn inform the design of novel HIV interventions. We have initiated a study to isolate, clone and functionally assess a genetically diverse panel of HIV-1 Vpu sequences for their CD4 and Tetherin downregulation abilities. Methods: HIV-1 Vpu protein sequences were isolated from plasma obtained from 207 ART naive HIV infected individuals from the UARTO cohort in Mbarara Uganda. Briefly, HIV RNA was extracted from plasma and nested RT-PCR was used to amplify the Vpu coding region. Amplicons were cloned into an expression vector, which features dual promotors driving Vpu and GFP expression, the latter as a marker of transfection efficiency. Vpu clones were transfected by electroporation into an immortalized CD4+ T-cell line (CEM). The ability of each clone to downregulate CD4 and Tetherin was quantified by flow cytometry and the resulting data normalized to that of negative (empty vector) and positive (Vpu from HIV reference strain NL4-3) controls. Results: To date, a total of 157 Vpu sequences have successfully been cloned and phylogenetically authenticated. Of these, 74 were HIV subtype D, 57 subtype A, 15 subtype C, and 11 recombinants (6 AD, 3 AE, 1 DE and 1 CD). A subset of 46 clones has been functionally assessed. Overall, normalized mean CD4 downregulation function of Vpu clones was 1.05 (standard deviation [SD] 0.37); normalized mean tetherin downregulation function was 0.85 [SD 0.15]). Notably, despite substantial genetic variability (in the form of single-nucleotide variants and insertions/deletions), our preliminary data do not reveal any significant functional differences in Vpu between HIV subtypes p=0.15 and p=0.71 for CD4 and Tetherin. Conclusion: This study will shed novel light on the extent to which global HIV-1 Vpu sequence diversity may impact the replicative and immune evasion functions of key viral accessory protein

POSTER #58

CD48 on B cells is crucial for their recognition and killing by effector CD8 T cells

Yu-Hsuan Huang 1,2, Kevin Tsai 1,2, Sohyeong Kang 1,2, Mandy L. Ford 3,

Marc S. Horwitz 4 and John J. Priatel 1,2

1 Childhood Diseases Theme, British Columbia Children’s Hospital Research Institute, 2 Department of

Pathology and Laboratory Medicine, University of British Columbia, 3 Department of Surgery, Emory University, 4 Department of Microbiology and Immunology, University of British Columbia

X-linked lymphoproliferative disease (XLP) is a rare primary immunodeficiency disease defined by exquisite sensitivity to the B-lymphotropic Epstein–Barr virus (EBV) and caused by inactivating mutations in the gene encoding SLAM-associated protein (SAP). SAP functions as an intracellular adaptor through association with surface SLAM family receptors and has been implicated in regulating lymphocyte/lymphocyte interactions. However, the precise mechanism of how the loss of SAP action on SLAM receptors contributes to extreme vulnerability to EBV remains unclear. Here, we hypothesize that SAP and SLAM family receptors are critical for cytotoxic effector CD8 T cell functions against virus-infected B cells. Importantly, we found that the expression of SAP and 2B4 by CD8 T cells is essential for their capacity to kill B cells but not other types of targets. Further, our results demonstrated that CD48 on the B cell surface is crucial for initiating 2B4-SAP-dependent signaling by effector CD8 T cells required for recognition and cytotoxicity of B cell targets both in vitro and in vivo. Altogether, our findings help explain the selective immune deficiency of XLP patients towards EBV by demonstrating the requirement of cytotoxic CD8 T cells on both SAP and 2B4 to kill B cells.

POSTER #59

The role of Lyn tyrosine kinase in monocyte biology and in immunotherapy regimes against metastatic lung cancer

Jessica Silva, Morgan Roberts, Maunish Barvalia, Kenneth Harder

Department of Microbiology and Immunology, University of British Columbia, Vancouver, British

Columbia, Canada. Emerging cancer immunotherapies are aimed at re-awaking the anti-tumour immune response by antagonizing receptor-ligand interactions responsible for suppressing anti-tumour T cell activity or selectively depleting immunosuppressive cells. Cancer immunotherapies have been shown to be clinically effective only in a minority of cancer patients, such as in metastatic melanoma, lymphoma, bladder and lung carcinomas. As such, strategies to improve patient responses and extend treatment regimes to other forms of cancer are required. Patrolling monocytes (pMo), have recently been identified as a key cell population involved in preventing tumour metastases to the lung. Additionally, pMos act as important regulators of tumour immunotherapies that depend on engagement of their Fc receptors by particular monoclonal antibody (mAb) isotypes, endowing these monocytes with the ability to selectively eliminate tumour cells or tumour stroma immunosuppressive cells. Data from our group shows that Lyn tyrosine kinase is a critical negative regulator of pMo development. We hypothesize that by selectively expanding pMos, tumour lung metastasis can be diminished and antibody-dependent depletion of tumour cells or immunosuppressive cells can be enhanced. We have developed metastatic melanoma and breast cancer cell lines in order to track pMo phagocytosis of tumour cells and tumour burden in the lungs of mice. Our in vivo results show that naïve Lyn-/- mice have increased numbers of circulating and lung pMos compared to WT mice. Lyn-/- mice also displayed a decreased metastatic melanoma lung tumour burden. By implementing a t-stochastic neighbour embedding algorithm on our flow cytometry data we were able to visualize phagocytosed mCherry-tumour cells by specific lung-resident myeloid populations including pMos. We propose that pMo expansion may decrease lung metastasis and enhance mAb-directed immunotherapy. We are exploring the mechanistic links between pMo number, function and immunotherapy efficacy using mass cytometry and tumour metastasis models with the goal of identifying new or synergistic cancer immunotherapy strategies.

POSTER #60

Disruption of the Gut Microbiota with Antibiotics Increases Acute Elucidating the role of Epstein-Barr virus infection in multiple sclerosis

using humanized mouse models of disease

Jessica R. Allanach1, Virginie S.E. Jean-Baptiste1, Iryna Shanina1, John J. Priatel2 and Marc S. Horwitz1

1Department of Microbiology & Immunology, University of British Columbia, Vancouver, Canada

2Department of Pathology & Laboratory Medicine, University of British Columbia, Vancouver, Canada Epstein-Barr virus (EBV), a human herpesvirus that establishes latent infection in B cells, is proposed to contribute to the pathogenesis of multiple sclerosis (MS) by mechanisms still unknown. Evidence for a role of EBV infection in MS comes from both epidemiological and experimental animal studies. Currently, there are no small animal models of MS that directly incorporate EBV due to its narrow tropism. Development of the peripheral blood mononuclear cell (PBMC) and bone marrow-liver-thymus (BLT) humanized mouse models has enabled infection of mice for the study of EBV-associated malignancies. To evaluate the role of latent EBV infection in MS, humanized mice were created by reconstitution of immunocompromised NSG mice with circulating human immune cells by one of two methods: engraftment of PBMCs from healthy EBV seropositive or seronegative donors (PBMC model), or by surgical implantation of fetal liver and thymus tissue under the kidney capsule followed by sub-lethal irradiation and injection of autologous CD34+ stem cells (BLT model). BLT mice were subsequently infected with EBV and infection status of the blood was tracked by quantitative PCR analysis. Experimental autoimmune encephalomyelitis (EAE) was induced in EBV-infected BLT and PBMC mice using recombinant human myelin oligodendrocyte glycoprotein (rhMOG). Preliminary experiments demonstrate humanized mice are susceptible to both EBV infection and EAE induction. PBMC mice showed differences in onset and incidence of EAE depending on the engrafted donor PBMCs and paralyzed mice showed significant T cell infiltration in the central nervous system, notably of IFNγ-expressing CD4 and CD8 T cells. Ongoing experiments are replicating these findings. Additionally, more donors are being recruited for analysis of EBV infection in the PBMC-EAE model, including MS patients.

POSTER #61

Homozygous MALT1 mutations are associated with impaired T cell signaling and effector functions.

Sohyeong Kang,1,2 Yu-Hsuan Huang,1,2 Kevin Tsai,1,2 Shan-Yu Fung,1,3 Stuart E.

Turvey,1,3 John J. Priatel1,2

1Childhood Diseases Theme, British Columbia Children’s Hospital Research Institute, 2Department of Pathology and Laboratory Medicine, University of British Columbia, 3Department of Pediatrics, University of

British Columbia Individuals with combined immunodeficiency exhibit near-normal T and B cell numbers but suffer from severe recurrent infections related to defective cellular or humoral immunity. Recently, a child presenting at BC Children’s Hospital with combined immunodeficiency was found to be homozygous for missense mutations in the MALT1 gene. MALT1 plays critical roles in NF-κB pathway upon T cell receptor stimulation in at least two different mechanisms: (1) as a scaffolding molecule bringing CARMA-1 and BCL-10 to form the CBM signalosome complex and (2) as a caspase-like protease (paracaspase) cleaving negative regulators of NF-κB signaling. Studies on Malt1−/− mice have revealed diminished NF-κB activation and weakened T cell immunity whereas mice lacking paracaspase activity with normal scaffolding function are prone to lethal autoimmunity. Together, these mouse findings raise questions regarding the nature of our patient’s mutant MALT1 protein and the role it plays in her proinflammatory phenotype. Our experiments have showed that the patient CD4 T cells display a normal frequency of regulatory T cells despite exhibiting impaired IL-2 production. In addition, we have found that the patient CD4 T cells exhibit defects in Th1 and Th17 differentiation and these impairments could be responsible for inefficient immune responses leading to chronic infections. Further, analyses of T cell signaling and paracaspase activity suggest that patient’s MALT1 mutation acts as a hypomorphic allele. Lastly, a better understanding of MALT1 function in T cells will provide invaluable knowledge of immune regulation and introduce MALT1-targeted therapies to treat T cell-mediated autoimmune diseases.

POSTER #62

Mucus deficiency results in negative selection of developing T cells restricted to oral antigens

Kevin Tsai, Yu-Hsuan Huang, Caixia Ma, Bruce A. Vallance & John J. Priatel

The Childhood Diseases Theme, British Columbia Children’s Hospital Research Institute and The Department of Pathology and Laboratory Medicine, The University of British Columbia, 950 West 28th Avenue, Vancouver,

BC V5Z 4H4 A thin layer of mucus functions to segregate contents of the intestinal lumen from the intestinal epithelium. MUC2 is the primary constituent of intestinal mucin and plays critical roles in the maintenance of immune cell homeostasis and protection against bacterial infection. Here, we investigate whether intestinal mucus is essential for maintaining T cell tolerance towards intestinal luminal antigens and preventing disease caused by constitutive T cell activation. To test this hypothesis, wild type and MUC2-deficient (Muc2-/-) mice were gavaged with the model antigen ovalbumin (OVA) and the elicited OVA-specific T cell responses monitored at various time points post-treatment. Significantly, the oral administration of OVA resulted in antigen being rapidly disseminated in the blood and lymphoid tissues of Muc2-/- but not wild type mice. Given the systemic spread of antigen in Muc2-/- mice, we next sought to investigate whether the selection of developing T cells in the thymus of Muc2-/- mice is impacted by dietary antigens. Remarkably, we found that gavage of Muc2-/- mice lead to presentation of OVA by thymic dendritic cells and the deletion of OVA-specific thymocytes whereas T cell development in wild type mice appeared unaffected. Importantly, our findings demonstrate that a leaky gut may result in intestinal luminal antigens shaping the T cell repertoire of developing thymocytes. Further, these results yield important insights into how T cell tolerance against intestinal luminal antigens is established and may lead to novel treatment strategies against allergies and autoimmune diseases.

POSTER #63

The IL-7 independent pathway of ILC2 development Maryam Ghaedi1,2, Abdalla Sheikh3, Itziar Martinez-Gonzalez1,2, Ninan Abraham3, and

Fumio Takei1,2

1Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC, Canada 2Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada 3Department of

Microbiology and Immunology, University of British Columbia, Vancouver, BC. Innate lymphoid cells (ILCs) are divided into group 1 (ILC1), group 2 (ILC2) and group 3 (ILC3). ILCs share a common precursor, ILC progenitor (ILCP), which expresses the transcription factor RORα. Allergic lung inflammation, also termed type 2 inflammation, is driven by overproduction of type 2 cytokines, including IL-5, and IL-13. Upon allergic encounter, a damaged lung epithelium releases alarmin IL-33 and activates ILC2s. Activated ILC2s produce large amounts of IL-5 and IL-13, which induce type 2 inflammation. As chronic type 2 inflammation can cause asthma, ILC2s play a critical role in asthma development. Single-cell RNA sequencing has indicated that ILC2s in the intestine is a heterogeneous population, which can be divided into subsets based on their cytokine and transcriptional profile. However, the existence of ILC2 subsets in lung tissue is not studied. To identify and characterize ILC2 subsets in the lung and in the context of allergic lung inflammation, I crossed RORα-IRES-Cre knock-in mice with Rosa26-floxSTOPflox-YFP mice to generate RORα/YFP mice, in which cells expressing RORα during development are permanently marked by YFP. RORα is expressed in ILC2s. Hence, all ILC2s are labeled by YFP. My preliminary analysis of these mice has shown that lung lineage-negative YFP+ cells include not only conventional ILC2s (CD127+CD25+ST2+) but also CD127−CD25+ST2+ cells. CD127−CD25+ST2+ cells produce IL-5 and IL-13 upon IL-33 stimulation, indicating that they are a subset of ILC2s. I also crossed RORα-IRES-Cre knock-in mice with IL-7Rfl mice to generate RORα/CD127 cKO mice. Analysis of RORα/CD127 cKO and CD127 KO mice indicates that small numbers of ILC2s can develop independently of CD127 signaling. This study provides new insights into ILC2 heterogeneity and the different developmental pathways involved in the generation of ILC2 subsets.

POSTER #64 Monitoring Respiratory Syncytial Virus Infections Using Whole Genome

Sequencing, Descriptive Epidemiology, and Viral Analysis Reveal the Transmission Dynamics and an Age Group Niche of RSV Types A and B

Elawar F.1, Bilawchuk L.1, Zhu D.1, Forss L.1, Hazes B.1, Jensen L.1, Griffiths C.1,

Drews S.2, Marchant D.1

1Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Alberta, Canada, 2Alberta Provincial Health Laboratory, PROVlab, Edmonton, Alberta, Canada

Background. The burden of Respiratory Syncytial Virus (RSV) infections in pediatric and elderly patients is recognized worldwide. However, transmission of RSV clades from season to season are poorly defined. Comprehensive monitoring systems are not well established for circulating strains in the community. Using bioinformatics data, this longitudinal study is a potential development in methods which predict strain persistence in the community. Methods. Clinical specimens from nasopharynx swabs were collected from patients with influenza like illnesses at clinics and hospitals in Alberta, Canada. Of the primary specimens, those that tested positive for RSV types-A or B, using Luminex, were submitted for further analysis from a larger ProvLab dataset. Samples were stratified: <1 year of age, between 1-10 years, and >60 years. The viral load from primary specimens was determined by quantitative RT-PCR and whole virus genomes were sequenced on an Illumina MiSeq platform. Phylogenetic trees were constructed for each RSV season we have monitored. This was done to determine circulating strains that carry over from the previous season. Foci of infection from the patient samples were titrated on Hep-2 cells, immunostained for RSV infection and analyzed by light-microscopy. We looked for correlations of in-vitro viral infection and patient viral load. All of these methods have been done for 3 monitored RSV seasons. Results. Viral loads of RSV-A and RSV-B in stratified age groups differed between the groups, suggesting an age group niche of RSV. These age group niches also differed each season suggesting there isn’t a consistency in RSV types A and B on which age group gets targeted. Independent of the strata, the size of foci of infection by viral isolates in Hep-2 cells correlated with viral load measured in patients. Phylogenetic trees identify strains persisting from year to year in the community. Phylogenetic data suggests virulent strains do not persist over time but strains which cause less serve disease evolve to acclimatize to the community. Conclusion. RSV type-A had an elevated viral load in children whereas RSV-type B was elevated in the elderly and infants in 2014. We determined a correlation between viral load in patients and the size of immunostained plaques in culture, implying that further bioinformatics work is needed to identify why strains are more virulent than others. We speculate SNPs may be involved in the viral virulence, and expect to determine a methodology for use on predictions for what types of strains will persist in the community.

POSTER #65

Systems biology provides mechanistic insights into the interaction of Aspergillus fumigatus conidia and human airway epithelial cells

Luka Culibrk1, Carys A. Croft1, Amreen Toor1,2, S. Jasemine Yang1,2, Gurpreet K. Singhera1, Delbert R. Dorscheid1,3, Margo M. Moore4 and Scott J. Tebbutt1,3,5

1: Centre for Heart Lung Innovation, University of British Columbia and St. Paul’s Hospital, Vancouver, Canada 2: Experimental Medicine, Department of Medicine, University of British Columbia, Vancouver,

Canada 3: Department of Medicine, Division of Respiratory Medicine, University of British Columbia, Vancouver, Canada 4: Department of Biological Sciences, Simon Fraser University, Burnaby, Canada 5: Prevention of Organ Failure (PROOF) Centre of Excellence, Vancouver, British Columbia, Canada

Introduction/Background: Aspergillus fumigatus is an opportunistic fungal pathogen causing often-fatal pulmonary infection in immunocompromised patients. One of the primary stages of infection by A. fumigatus spores (conidia) is interaction of inhaled conidia with airway epithelial cells. Previously, we have demonstrated that human airway epithelial cells internalize A. fumigatus conidia in cell culture models and it is hypothesized that this could provide a means by which inhaled conidia evade the innate immune system of the host. By applying the vast wealth of available public data in a systems biology approach, we aimed to further investigate the interaction of A. fumigatus conidia with human airway epithelial cells. Methods/Results: Using transcriptome-wide data previously generated in our laboratory as well as public data available in the sequence read archive, we identified the human gene WAS-interacting protein family member 2 (WIPF2) as a candidate gene of interest in the interaction of A. fumigatus conidia with human airway epithelial cells. Using RNAi and the nystatin protection assay, we demonstrated that knockdown of WIPF2 in an airway epithelial cell culture model reduced internalization of A. fumigatus conidia by over two-fold. Finally, we used immunofluorescence-confocal microscopy to demonstrate co-localization of WIPF2 to A. fumigatus conidia during early stages of infection. Discussion/Significance: Identification and validation of WIPF2 in our study significantly improves onf our current models of A. fumigatus interaction with the lung epithelium. Previously, our knowledge of how airway epithelial cells internalize conidia was limited to the knowledge that actin played a role in this process. WIPF2 is already known to regulate the rearrangement of the actin cytoskeleton by activation of the Arp2/3 complex. These findings are likely to promote the investigation of WIPF2, the Arp2/3 complex and other WIPF2-interacting proteins in this and other host-pathogen systems and represents a clear case for the use of systems biology to investigate host-pathogen interactions.

POSTER #66

Fast and Easy Immunomagnetic Positive Selection of PE- or Biotin-Conjugated Antibody Labeled Cells with Releasable RapidSpheres™

Results in Highly Purified, Functional and Particle-Free Cells

Catherine L. Ewen1, Andy I. Kokaji1, Samuel J. Clarke1, Ulrike Lambertz1, Susan de Jong1, Steven M. Woodside1, Terry E. Thomas1, and Allen C. Eaves1,2

1 STEMCELL Technologies Inc., Vancouver, BC, Canada 2 Terry Fox Laboratory, BC Cancer Agency,

Vancouver, BC, Canada Immunomagnetic cell isolation is a cost-effective and gentle method of obtaining purified cell populations. However, isolating novel or rare cell populations often relies on relatively time consuming and expensive cell sorting techniques due to the lack of off-the-shelf immunomagnetic cell isolation kits. To bridge this gap, we have developed the EasySep™ Release isolation kits for PE- or biotin-conjugated antibodies. EasySep™ Release uses magnetic particles with low non-specific binding characteristics that can be rapidly removed from isolated cells after positive selection. The system is highly customizable, targeting cells through the use of any biotin- or PE-conjugated primary antibody or ligand. Starting with a variety of sample materials, including single-cell suspensions from mouse spleen, lymph nodes, lungs, rat spleen, or human peripheral blood mononuclear cells (PBMCs), the EasySep™ Release kits have consistently demonstrated purities above 85%. The EasySep™ Release technology can also be used to isolate subsets of cells when used in combination with our other cell separation products. For example, functional and particle-free mouse CD25+ or CD304+CD4+ regulatory T cells were isolated by using our EasySep™ Release Mouse PE Positive Selection Kit and EasySep™ Mouse CD4+ T Cell Isolation Kit. We have used EasySep™ Release Mouse kits with EasySep™ Dextran RapidSpheres™ to isolate CD4+CD8+ mouse thymocytes (double positive selection) and to isolate mouse lung epithelial cells by EpCAM positive selection, followed by the removal of contaminating leukocytes via CD45 depletion. These kits offer a customizable and cost-effective magnetic isolation approach for the isolation of virtually any cell population.

POSTER #67

Elucidating the epigenetic regulation of CD4+ T cell derived IL-10 during influenza infection

Nicolette Fonseca1, Alireza Lorzadeh2, Stephen A. Redpath1, Martin Hirst2 and

Georgia Perona Wright1

1Department of Microbiology and Immunology; 2Centre for High Throughput Biology, Department of

Microbiology and Immunology, University of British Columbia, Vancouver, Canada.

Surviving influenza requires a precise balance of pro-inflammatory signals that promote viral clearance and anti-inflammatory signals that prevent immunopathology. IL-10 is a potent immunosuppresive cytokine that is critical to this balance. Evidence suggests that in CD4+ T cells, IL-10 expression is dependent on IL-27 signaling. Our ex vivo analysis of CD4+ T cell IL-10 expression shows that IL-10 is indeed dependent on IL-27 signaling in a primary response to influenza however in a recall response, CD4+ T cells express IL-10 in an IL-27 independent manner. Previous studies have shown that T cell gene expression can be epigenetically regulated through histone modifications. We hypothesized that the Il10 gene locus in memory CD4+ T cells may be remodeled to an open state following primary infection. To assess the correlation between permissive histone modification patterns and memory CD4+ T cell IL-10 expression, we performed native ChIP-Seq on aliquots of ~10,000 flow-sorted naïve and influenza-specific memory CD4+ T cells. Four histone modifications (H3K4me3, H3K4me1, H3K27me3, and H3K27ac) were profiled with the ChIP Seq analysis tool MACS2. We observed that memory CD4+ T cells acquire permissive H3K4me3, H3K4me1 and H3K27Ac signatures and lose the repressive H3K27me3 mark at the Il10 locus compared to naïve CD4 T cells. Together our data suggest that despite dependence on IL-27 in a primary response, memory CD4+ T cells acquire a permissive epigenetic signature at the Il10 locus, which positively correlates with their ability to express IL-10 in a recall response to influenza.